scholarly journals An Inhibitory Role of Osthole in Rat MSCs Osteogenic Differentiation and Proliferation via Wnt/β-Catenin and Erk1/2-MAPK Pathways

2016 ◽  
Vol 38 (6) ◽  
pp. 2375-2388 ◽  
Author(s):  
Hongyang Hu ◽  
Min Chen ◽  
Guangzu Dai ◽  
Guoqing Du ◽  
Xuezong Wang ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Signaling Pathways ◽  
Western Blotting ◽  
Underlying Mechanism ◽  
Mapk Signaling ◽  
Cell Counting ◽  
Nuclear Antigen ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Mapk Signaling Pathways

Background/Aims: Bone marrow-derived mesenchymal stem cells (MSCs) are responsible for new bone formation during adulthood. Accumulating evidences showed that Osthole promotes the osteogenic differentiation in primary osteoblasts. The aim of this study was to investigate whether Osthole exhibits a potential to stimulate the osteogenic differentiation of MSCs and the underlying mechanism. Methods: MSCs were treated with a gradient concentration of Osthole (6.25 µM, 12.5 µM, and 25 µM). Cell proliferation was assessed by western blotting with the proliferating cell nuclear antigen (PCNA) and Cyclin D1 antibodies, fluorescence activated cell sorting (FACS), and cell counting kit 8 (CCK8). MSCs were cultured in osteogenesis-induced medium for one or two weeks. The osteogenic differentiation of MSCs was estimated by Alkaline Phosphatase (ALP) staining, Alizarin red staining, Calcium influx, and quantitative PCR (qPCR). The underlying mechanism of Osthole-induced osteogenesis was further evaluated by western blotting with antibodies in Wnt/β-catenin, PI3K/Akt, BMPs/smad1/5/8, and MAPK signaling pathways. Results: Osthole inhibited proliferation of rat MSCs in a dose-dependent manner. Osthole suppressed osteogenic differentiation of rat MSCs by down-regulating the activities of Wnt/β-catenin and Erk1/2-MAPK signaling. Conclusions: Osthole inhibits the proliferation and osteogenic differentiation of rat MSCs, which might be mediated through blocking the Wnt/β-catenin and Erk1/2-MAPK signaling pathways.

scholarly journals Expression and Secretion of Cathelicidin LL-37 in Human Epithelial Cells after Infection by Mycobacterium bovis Bacillus Calmette-Guérin

2008 ◽  
Vol 15 (9) ◽  
pp. 1450-1455 ◽  
Author(s):  
Patricia Méndez-Samperio ◽  
Elena Miranda ◽  
Artemisa Trejo
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Mrna Expression ◽  
Signaling Pathways ◽  
Western Blotting ◽  
Mycobacterium Bovis ◽  
A549 Cells ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Mapk Signaling Pathways

ABSTRACT The antimicrobial cathelicidin LL-37 is considered to play an important role in the innate immune response to tuberculosis infection. However, little is known about the induction and secretion of this antimicrobial peptide in A549 epithelial cells after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), the world's most widely used tuberculosis vaccine. In this study, we investigated the effect of M. bovis BCG on LL-37 mRNA levels in A549 cells by real-time PCR and on protein levels by Western blotting. Treatment of cells with M. bovis BCG upregulates LL-37 mRNA expression in a dose- and time-dependent manner. The quantitative analysis of LL-37 gene expression correlated with our Western blotting results. Moreover, our results demonstrated that treatment of cells with the transcriptional inhibitor actinomycin D effectively inhibited in a concentration-dependent manner the ability of M. bovis BCG to induce LL-37 mRNA expression. Finally, inhibition of the MEK1/2 and p38 mitogen-activated protein kinase (MAPK) signaling pathways reduced M. bovis BCG-mediated LL-37 mRNA expression, a reduction that correlated with the observed high level of downregulation of LL-37 protein induction. Thus, these results indicate that the MEK1/2 and p38 MAPK signaling pathways play a critical role in the regulation of inducible LL-37 gene expression in A549 cells infected with M. bovis BCG.

scholarly journals Halofuginone Sensitizes Lung Cancer Organoids to Cisplatin via Suppressing PI3K/AKT and MAPK Signaling Pathways

2021 ◽  
Vol 9 ◽  
Author(s):  
Hefei Li ◽  
Yushan Zhang ◽  
Xiaomei Lan ◽  
Jianhua Yu ◽  
Changshuang Yang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Underlying Mechanism ◽  
Mapk Signaling ◽  
Lung Cancer Cells ◽  
Dependent Manner ◽  
New Strategy ◽  
Resistant Patient ◽  
Mapk Signaling Pathways

Lung cancer is the leading cause of cancer death worldwide. Cisplatin is the major DNA-damaging anticancer drug that cross-links the DNA in cancer cells, but many patients inevitably develop resistance with treatment. Identification of a cisplatin sensitizer might postpone or even reverse the development of cisplatin resistance. Halofuginone (HF), a natural small molecule isolated from Dichroa febrifuga, has been found to play an antitumor role. In this study, we found that HF inhibited the proliferation, induced G0/G1 phase arrest, and promoted apoptosis in lung cancer cells in a dose-dependent manner. To explore the underlying mechanism of this antitumor effect of halofuginone, we performed RNA sequencing to profile transcriptomes of NSCLC cells treated with or without halofuginone. Gene expression profiling and KEGG analysis indicated that PI3K/AKT and MAPK signaling pathways were top-ranked pathways affected by halofuginone. Moreover, combination of cisplatin and HF revealed that HF could sensitize the cisplatin-resistant patient-derived lung cancer organoids and lung cancer cells to cisplatin treatment. Taken together, this study identified HF as a cisplatin sensitizer and a dual pathway inhibitor, which might provide a new strategy to improve prognosis of patients with cisplatin-resistant lung cancer.

scholarly journals MEK1/2 Inhibitor (GDC0623) Promotes Osteogenic Differentiation of Primary Osteoblasts Inhibited by IL-1β through the MEK-Erk1/2 and Jak/Stat3 Pathways

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Zeng-Qiao Zhang ◽  
Xiao-Shen Hu ◽  
Ye-Chen Lu ◽  
Jun-Peng Zhang ◽  
Wen-Yao Li ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Western Blotting ◽  
Cell Counting ◽  
Nuclear Antigen ◽  
Control Group ◽  
Induction Medium ◽  
Alizarin Red ◽  
Real Time Quantitative Pcr ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction

Objective. We evaluated the effects and mechanisms of GDC0623 on osteogenic differentiation of osteoblasts induced by IL-1β. Methodology. Osteoblasts were treated with 20 ng/ml IL-1β and 0.1 µM GDC0623. Cell proliferation levels were evaluated by the cell counting kit 8 (CCK8), EdU assay, and western blotting [proliferating cell nuclear antigen (PCNA) and Cyclin D1]. Osteoblasts were cultured in an osteogenic induction medium for 1–3 weeks after which their differentiations were assessed by alkaline phosphatase (ALP) staining, Alizarin Red staining, calcium concentration, immunocytochemistry staining, real-time quantitative PCR (RT-qPCR), and immunofluorescence staining. The osteogenesis-associated mechanisms were further evaluated by western blotting using appropriate antibodies. Results. Relative to the control group, IL-1β induced the rapid proliferation of osteoblasts and suppressed their osteogenic differentiations by upregulating the activities of MEK-Erk1/2 as well as Jak-Stat3 pathways and by elevating MMP13 and MMP9 levels. However, blocking of the MEK-Erk1/2 signaling pathway by GDC0623 treatment reversed these effects. Conclusion. Inhibition of Jak-Stat3 pathway by C188-9 downregulated the expression levels of MMP9 and MMP13, activated MEK-Erk1/2 pathway, and inhibited osteogenic differentiation.

scholarly journals Yunnan Baiyao Conditioned Medium Promotes the Odonto/Osteogenic Capacity of Stem Cells from Apical Papilla via Nuclear Factor Kappa B Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Xiyao Pang ◽  
Yanqiu Wang ◽  
Jintao Wu ◽  
Zhou Zhou ◽  
Tao Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Conditioned Medium ◽  
Signaling Pathway ◽  
Cell Counting ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Apical Papilla ◽  
Yunnan Baiyao

Yunnan Baiyao is a traditional Chinese herbal remedy that has long been used for its characteristics of wound healing, bone regeneration, and anti-inflammation. However, the effects of Yunnan Baiyao on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) and the potential mechanisms remain unclear. The aim of this study was to investigate the odonto/osteogenic differentiation effects of Yunnan Baiyao on SCAPs and the underlying mechanisms involved. SCAPs were isolated and cocultured with Yunnan Baiyao conditioned media. The proliferation ability was determined by cell counting kit 8 and flow cytometry. The differentiation capacity and the involvement of NF-κB pathway were investigated by alkaline phosphatase assay, alizarin red staining, immunofluorescence assay, real-time RT-PCR, and western blot analyses. Yunnan Baiyao conditioned medium at the concentration of 50 μg/mL upregulated alkaline phosphatase activity, induced more mineralized nodules, and increased the expression of odonto/osteogenic genes/proteins (e.g., OCN/OCN, OPN/OPN, OSX/OSX, RUNX2/RUNX2, ALP/ALP, COL-I/COL-I, DMP1, DSP/DSPP) of SCAPs. In addition, the expression of cytoplasmic phos-IκBα, phos-P65, and nuclear P65 was significantly increased in Yunnan Baiyao conditioned medium treated SCAPs in a time-dependent manner. Conversely, the differentiation of Yunnan Baiyao conditioned medium treated SCAPs was obviously inhibited when these stem cells were cocultured with the specific NF-κB inhibitor BMS345541. Yunnan Baiyao can promote the odonto/osteogenic differentiation of SCAPs via the NF-κB signaling pathway.

scholarly journals The effects of 50 Hz magnetic field–exposed cell culture medium on cellular functions in FL cells

2019 ◽  
Vol 60 (4) ◽  
pp. 424-431 ◽  
Author(s):  
Yue Fei ◽  
Liling Su ◽  
Haifeng Lou ◽  
Chuning Zhao ◽  
Yiqin Wang ◽  
...  
Keyword(s):  
Cell Viability ◽  
Signaling Pathways ◽  
Relative Permittivity ◽  
Culture Medium ◽  
Biological Effects ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Cell Counting ◽  
International Agency ◽  
Dependent Manner

Abstract Although extremely low frequency magnetic fields (ELF-MFs) have been classified as a possible carcinogen for humans by the International Agency for Research on Cancer (IARC), their biological effects and underlying mechanisms are still unclear. Our previous study indicated that ELF-MF exposure influenced the relative permittivity of the saline solution, suggesting that the MF exposure altered physical properties of the solution. To explore the biophysical mechanism of ELF-MF–induced biological effects, this study examined the effects of 50 Hz sinusoidal MF at 0–4.0 mT on the permittivity of culture medium with phase-interrogation surface plasmon resonance (SPR) sensing. Then, the biological effects of MF pre-exposed culture medium on cell viability, the mitogen-activated protein kinase (MAPK) signaling pathways, oxidative stress, and genetic stabilities were analyzed using Cell Counting Kit-8, western blot, flow cytometry, γH2AX foci formation, and comet assay. The results showed that SPR signals were decreased under MF exposure in a time- and dose-dependent manner, and the decreased SPR signals were reversible when the exposure was drawn off. However, MF pre-exposed culture medium did not significantly change cell viability, intracellular reactive oxygen species level, activation of the MARK signaling pathways, or genetic stabilities in human amniotic epithelial cells (FL cells). In conclusion, our data suggest that the relative permittivity of culture medium was influenced by 50 Hz MF exposure, but this change did not affect the biological processes in FL cells.

Berberine inhibits lipopolysaccharide-induced expression of inflammatory cytokines by suppressing TLR4-mediated NF-ĸB and MAPK signaling pathways in rumen epithelial cells of Holstein calves

2019 ◽  
Vol 86 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Chenxu Zhao ◽  
Yazhou Wang ◽  
Xue Yuan ◽  
Guoquan Sun ◽  
Bingyu Shen ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Signaling Pathways ◽  
Inflammatory Cytokines ◽  
Molecular Mechanisms ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Inflammatory Drug ◽  
Anti Inflammatory ◽  
Mapk Signaling Pathways ◽  
Rumen Epithelial Cells

AbstractSubacute ruminal acidosis (SARA) can increase the level of inflammation and induce rumenitis in dairy cows. Berberine (BBR) is the major active component of Rhizoma Coptidis, which is a type of Chinese anti-inflammatory drug for gastrointestinal diseases. The purpose of this study was to investigate the anti-inflammatory effects of BBR on lipopolysaccharide (LPS)-stimulated rumen epithelial cells (REC) and the underlying molecular mechanisms. REC were cultured and stimulated with LPS in the presence or absence of different concentrations of BBR. The results showed that cell viability was not affected by BBR. Moreover, BBR markedly decreased the concentrations and mRNA expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the LPS-treated REC in a dose-dependent manner. Importantly, Western blotting analysis showed that BBR significantly suppressed the protein expression of toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein (MyD88) and the phosphorylation of nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) in LPS-treated REC. Furthermore, the results of immunocytofluorescence showed that BBR significantly inhibited the nuclear translocation of NF-κB p65 induced by LPS treatment. In conclusion, the protective effects of BBR on LPS-induced inflammatory responses in REC may be due to its ability to suppress the TLR4-mediated NF-κB and MAPK signaling pathways. These findings suggest that BBR can be used as an anti-inflammatory drug to treat inflammation induced by SARA.

MAPK signaling pathways are needed for survival of H9c2 cardiac myoblasts under extracellular alkalosis

2008 ◽  
Vol 295 (3) ◽  
pp. H1319-H1329 ◽  
Author(s):  
Konstantina Stathopoulou ◽  
Isidoros Beis ◽  
Catherine Gaitanaki
Keyword(s):  
Signaling Pathways ◽  
P38 Mapk ◽  
Cardiac Myocyte ◽  
Consensus Sequence ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Selective Inhibitors ◽  
Cardiac Myoblasts ◽  
Mapk Signaling Pathways

pH is one of the most important physiological parameters, with its changes affecting the function of vital organs like the heart. However, the effects of alkalosis on the regulation of cardiac myocyte function have not been extensively investigated. Therefore, we decided to study whether the mitogen-activated protein kinase (MAPK) signaling pathways [c-Jun NH2-terminal kinases (JNKs), extracellular signal-regulated kinases (ERKs), and p38 MAPK] are activated by alkalosis induced with Tris-Tyrode buffer at two pH values, 8.5 and 9.5, in H9c2 rat cardiac myoblasts. These buffers also induced intracellular alkalinization comparable to that induced by 1 mM NH4Cl. The three MAPKs examined presented differential phosphorylation patterns that depended on the severity and the duration of the stimulus. Inhibition of Na+/H+ exchanger (NHE)1 by its inhibitor HOE-642 prevented alkalinization and partially attenuated the alkalosis (pH 8.5)-induced activation of these kinases. The same stimulus also promoted c-Jun phosphorylation and enhanced the binding at oligonucleotides bearing the activator protein-1 (AP-1) consensus sequence, all in a JNK-dependent manner. Additionally, mitogen- and stress-activated kinase 1 (MSK1) was transiently phosphorylated by alkalosis (pH 8.5), and this was abolished by the selective inhibitors of either p38 MAPK or ERK pathways. JNKs also mediated Bcl-2 phosphorylation in response to incubation with the alkaline medium (pH 8.5), while selective inhibitors of the three MAPKs diminished cell viability under these conditions. All these data suggest that alkalosis activates MAPKs in H9c2 cells and these kinases, in turn, modify proteins that regulate gene transcription and cell survival.

scholarly journals The Role of Pannexin3-Modified Human Dental Pulp-Derived Mesenchymal Stromal Cells in Repairing Rat Cranial Critical-Sized Bone Defects

2017 ◽  
Vol 44 (6) ◽  
pp. 2174-2188 ◽  
Author(s):  
Fangfang Song ◽  
Hualing Sun ◽  
Liyuan Huang ◽  
Dongjie Fu ◽  
Cui Huang
Keyword(s):  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Dental Pulp ◽  
Underlying Mechanism ◽  
Bone Defects ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Human Dental Pulp

Background/Aims: Human dental pulp-derived mesenchymal stromal cells (hDPSCs) are promising seed cells for tissue engineering due to their easy accessibility and multi-lineage differentiation. Pannexin3 (Panx3) plays crucial roles during bone development and differentiation. The aim of the present study was to investigate the effect of Panx3 on osteogenesis of hDPSCs and the underlying mechanism. Methods: Utilizing qRT-PCR, Western blot, and immunohistochemistry, we explored the change of Panx3 during osteogenic differentiation of hDPSCs. Next, hDPSCs with loss (Panx3 knockdown) and gain (Panx3 overexpression) of Panx3 function were developed to investigate the effects of Panx3 on osteogenic differentiation of hDPSC and the underlying mechanism. Finally, a commercial β-TCP scaffold carrying Panx3-modified hDPSCs was utilized to evaluate bone defect repair. Results: Panx3 was upregulated during osteogenic differentiation in a time-dependent manner. Panx3 overexpression promoted osteogenic differentiation of hDPSCs, whereas depletion of Panx3 resulted in a decline of differentiation, evidenced by upregulated expression of mineralization-related markers, increased alkaline phosphatase (ALP) activity, and enhanced ALP and Alizarin red staining. Panx3 was found to interact with the Wnt/β-catenin signaling pathway, forming a negative feedback loop. However, Wnt/β-catenin did not contribute to enhancement of osteogenic differentiation as observed in Panx3 overexpression. Moreover, Panx3 promoted osteogenic differentiation of hDPSCs via increasing ERK signaling pathway. Micro-CT and histological staining results showed that Panx3-modified hDPSCs significantly improved ossification of critical-sized bone defects. Conclusion: These findings suggest that Panx3 is a crucial modulator of hDPSCs differentiation.

scholarly journals Hsp90 Modulates Human Sperm Capacitation via the Erk1/2 and p38 MAPK Signaling Pathways

2020 ◽  
Author(s):  
Peibei Sun ◽  
Yayan Wang ◽  
Tian Gao ◽  
Kun Li ◽  
Dongwang Zheng ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
P38 Mapk ◽  
Sperm Motility ◽  
Western Blotting ◽  
Protein Complex ◽  
Acrosome Reaction ◽  
Human Sperm ◽  
Mapk Signaling ◽  
Sperm Capacitation ◽  
Mapk Signaling Pathways

Abstract Background: Heat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction. Previously, we found that both Hsp90 and its co-chaperone cell division cycle protein 37 (Cdc37) were expressed in human sperm. Hsp90 is known to be involved in human sperm capacitation via unknown underlying mechanism(s). As Cdc37 was a kinase-specific co-chaperone of Hsp90, Hsp90 may regulate human sperm capacitation via other kinases. It has been reported that two major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38, are expressed in human sperm in the same locations as Hsp90 and Cdc37. Phosphorylated Erk1/2 has been shown to promote sperm hyperactivated motility and acrosome reaction, while phosphorylated p38 inhibits sperm motility. Therefore, in this study we explored whether Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways. Methods: Human sperm was treated with the Hsp90-specific inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) during capacitation. Computer-assisted sperm analyzer (CASA) was used to detect sperm motility and hyperactivation. The sperm acrosome reaction was analyzed by using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC) staining. The interactions between Hsp90, Cdc37, Erk1/2 and p38 were assessed using co-immunoprecipitation (Co-IP) experiments. Western blotting analysis was used to evaluate the levels of protein expression and phosphorylation. Results: Human sperm hyperactivation and acrosome reaction were inhibited by 17-AAG, suggesting that Hsp90 is involved in human sperm capacitation. In addition, Co-IP experiments revealed that 17-AAG reduced the interaction between Hsp90 and Cdc37, leading to the dissociation of Erk1/2 from the Hsp90-Cdc37 protein complex. Western blotting analysis revealed that levels of Erk1/2 and its phosphorylated form were subsequently decreased. Decreasing of Hsp90-Cdc37 complex also affected the interaction between Hsp90 and p38. Nevertheless, p38 dissociated from the Hsp90 protein complex and was activated by autophosphorylation. Conclusions: Taken together, our findings indicate that Hsp90 is involved in human sperm hyperactivation and acrosome reaction. In particular, Hsp90 and its co-chaperone Cdc37 form a protein complex with Erk1/2 and p38 to regulate their kinase activity. These results suggest that Hsp90 regulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.

scholarly journals Immune activation of murine RAW264.7 macrophages by sonicated and alkalized paramylon from Euglena gracilis

2019 ◽  
Author(s):  
Qingqing Guo ◽  
Decheng Bi ◽  
Mingcan Wu ◽  
Boming Yu ◽  
Lang Hu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Immune System ◽  
Signaling Pathways ◽  
Euglena Gracilis ◽  
Mapk Signaling ◽  
Regulation Mechanism ◽  
Dependent Manner ◽  
Raw264.7 Macrophages ◽  
Therapeutic Benefits ◽  
Mapk Signaling Pathways

Abstract Background: Euglena as a new super health food resource that is rich in the natural polysaccharide paramylon, a linear β-1,3-glucan with various biological activities including activity on the immune system in different cell lines and animals. Despite these reports, the immune regulation mechanism of paramylon is still unclear. Results: We investigate the signaling pathways paramylon impacts in immune macrophages. In RAW264.f:7 macrophages, sonicated and alkalized paramylon oligomers up-regulated inducible nitric oxide synthase (iNOS) and increased secretion of nitric oxide (NO), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in a concentration-dependent manner. In addition, paramylon activated the nuclear factor-κB(NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and inhibiting these pathways attenuated the paramylon-induced secretion of the above immune-mediators. Conclusions: These results demonstrate that Euglena gracilis paramylon modulates the immune system via activation of the NF-κB and MAPK signaling pathways and thus has potential therapeutic benefits.

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