scholarly journals N-Cadherin Attenuates High Glucose-Induced Nucleus Pulposus Cell Senescence Through Regulation of the ROS/NF-κB Pathway

2018 ◽  
Vol 47 (1) ◽  
pp. 257-265 ◽  
Author(s):  
Gang Hou ◽  
Huiqing Zhao ◽  
Haijun Teng ◽  
Pei Li ◽  
Wenbin Xu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nucleus Pulposus ◽  
Western Blotting ◽  
High Glucose ◽  
Culture Medium ◽  
Telomerase Activity ◽  
Cell Senescence ◽  
Cell Phenotype ◽  
P53 Expression ◽  
Pathway Activity

Background/Aims: Diabetes mellitus (DM) is a potential etiology of disc degeneration. N-cadherin (N-CDH) helps maintain the cell viability, cell phenotype and matrix biosynthesis of nucleus pulposus (NP) cells. Here, we mainly aimed to investigate whether N-CDH can attenuate high glucose-induced NP cell senescence and its potential mechanism. Methods: Rat NP cells were cultured in a base culture medium and base culture medium with a 0.2 M glucose concentration. Recombinant lentiviral vectors were used to enhance N-CDH expression in NP cells. Senescence-associated β-galactosidase (SA-β-Gal) activity was measured by SA-β-Gal staining. NP cell proliferation was evaluated by CCK-8 assay. Telomerase activity and intracellular reactive oxygen species (ROS) content were tested by specific chemical kits according to the manufacturer’s instructions. G0/G1 cell cycle arrest was evaluated by flow cytometry. Real-time PCR and Western blotting were used to analyze mRNA and protein expressions of senescence markers (p16 and p53) and matrix macromolecules (aggrecan and collagen II). Additionally, p-NF-κB expression was also analyzed by Western blotting to evaluate NF-κB pathway activity. Results: High glucose significantly decreased N-CDH expression, increased ROS generation and NF-κB pathway activity, and promoted NP cell senescence, which was reflected in the increase in SA-β-Gal activity and senescence marker (p16 and p53) expression, compared to the control group. High glucose decreased telomerase activity and cell proliferation potency. However, N-CDH overexpression partially attenuated NP cell senescence, decreased ROS content and inhibited the activation of the NF-κB pathway under the high glucose condition. Conclusion: High glucose decreases N-CDH expression and promotes NP cell senescence. N-CDH overexpression can attenuate high glucose-induced NP cell senescence through the regulation of the ROS/ NF-κB pathway. This study suggests that N-CDH is a potential therapeutic target to slow DM-mediated disc NP degeneration.

scholarly journals Hyper-osmolarity environment-induced oxidative stress injury promotes nucleus pulposus cell senescence in vitro

2019 ◽  
Vol 39 (9) ◽  
Author(s):  
Jiawei Xu ◽  
Haopeng Li ◽  
Kai Yang ◽  
Shuai Guo ◽  
Jie Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Proliferation ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Telomerase Activity ◽  
Cell Senescence ◽  
Stress Injury ◽  
Regulated Expression ◽  
Collagen Ii

Abstract Nucleus pulposus (NP) cell senescence is involved in disc degeneration. The in situ osmolarity within the NP region is an important regulator of disc cell’s biology. However, its effects on NP cell senescence remain unclear. The present study was aimed to investigate the effects and mechanism of hyper-osmolarity on NP cell senescence. Rat NP cells were cultured in the in situ-osmolarity medium and hyper-osmolarity medium. The reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) was added along with the medium to investigate the role of oxidative injury. Cell cycle, cell proliferation, senescence associated β-galactosidase (SA-β-Gal) activity, telomerase activity, expression of senescence markers (p16 and p53) and matrix molecules (aggrecan and collagen II) were tested to assess NP cell senescence. Compared with the in situ-osmolarity culture, hyper-osmolarity culture significantly decreased cell proliferation and telomerase activity, increased SA-β-Gal activity and cell fraction in the G0/G1 phase, up-regulated expression of senescence markers (p16 and p53) and down-regulated expression of matrix molecules (aggrecan and collagen II), and increased intracellular ROS accumulation. However, addition of NAC partly reversed these effects of hyper-osmolarity culture on cellular senescence and decreased ROS content in NP cells. In conclusion, a hyper-osmolarity culture promotes NP cell senescence through inducing oxidative stress injury. The present study provides new knowledge on NP cell senescence and helps us to better understand the mechanism of disc degeneration.

scholarly journals Optimization of Spheroid Colony Culture and Cryopreservation of Nucleus Pulposus Cells for the Development of Intervertebral Disc Regenerative Therapeutics

2021 ◽  
Vol 11 (8) ◽  
pp. 3309
Author(s):  
Kosuke Sako ◽  
Daisuke Sakai ◽  
Yoshihiko Nakamura ◽  
Erika Matsushita ◽  
Jordy Schol ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Viability ◽  
Nucleus Pulposus ◽  
Mass Culture ◽  
Formation Rate ◽  
Proliferation Rate ◽  
Cell Phenotype ◽  
Mrna Levels ◽  
Spheroid Formation ◽  
Positive Nucleus

After the discovery of functionally superior Tie2-positive nucleus pulposus (NP) progenitor cells, new methods were needed to enable mass culture and cryopreservation to maintain these cells in an undifferentiated state with high cell yield. We used six types of EZSPHERE® dishes, which support spheroid-forming colony culture, and examined NP cell spheroid-formation ability, number, proliferation, and mRNA expression of ACAN, COL1A2, COL2A1, and ANGPT1. Six different types of cryopreservation solutions were examined for potential use in clinical cryopreservation by comparing the effects of exposure time during cryopreservation on cell viability, Tie2-positivity, and cell proliferation rates. The spheroid formation rate was 45.1% and the cell proliferation rate was 7.75 times using EZSPHERE® dishes. The mRNA levels for COL2A1 and ANGPT1 were also high. In cryopreservation, CryoStor10 (CS10) produced ≥90% cell viability and a high proliferation rate after thawing. CS10 had a high Tie2-positive rate of 12.6% after culturing for 5 days after thawing. These results suggest that EZSPHERE enabled colony formation in cell culture without the use of hydrogel products and that CS10 is the best cryopreservation medium for retaining the NP progenitor cell phenotype and viability. Together, these data provide useful information of NP cell-based therapeutics to the clinic.

scholarly journals Mechanical Stretch Induces Annulus Fibrosus Cell Senescence through Activation of the RhoA/ROCK Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Li Ning ◽  
Lei Gao ◽  
Fan Zhang ◽  
Xiaoxiao Li ◽  
Tingting Wang
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Annulus Fibrosus ◽  
Coiled Coil ◽  
Mechanical Stretch ◽  
Telomerase Activity ◽  
Cell Senescence ◽  
Central Nucleus ◽  
Biological Behavior ◽  
Signaling Transduction Pathway

Background. Intervertebral disc is responsible for absorbing and transmitting mechanical compression. Under physiological conditions, the peripheral annulus fibrosus (AF) cells are subjected to different magnitudes of transverse mechanical stretch depending on the swelling of the central nucleus pulposus tissue. However, the biological behavior of AF cells under mechanical stretch is not well studied. Objective. This study was performed to study the effects of mechanical tension on AF cell senescence and the potential signaling transduction pathway. Methods. Rat AF cells were made to experience different magnitudes of mechanical stretch (2% elongation and 20% elongation for 4 hours every day at 1 Hz) in a 10-day experiment period. The inhibitor RKI-1447 of the Rho-associated coiled-coil–containing protein kinases (ROCK) was added along with culture medium to investigate its role. Cell proliferation, cell cycle, telomerase activity, and expression of senescence markers (p16 and p53) were analyzed. Results. We found that 20% elongation significantly decreased cell proliferation, promoted G0/G1 cell cycle arrest, decreased telomerase activity, and upregulated mRNA/protein expression of p16 and p53. Moreover, the inhibitor RKI-1447 partly resisted effects of 20% elongation on these parameters of cell senescence. Conclusion. High mechanical stretch obviously induces AF cell senescence through the RhoA/ROCK pathway. This study provides us a deeper understanding on the AF cell’s behavior under mechanical stretch.

Bimodal effects of platelet-derived growth factor on rat mesangial cell proliferation and death, and the role of lysophosphatidic acid in cell survival

2001 ◽  
Vol 101 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Chiyoko N. INOUE ◽  
Isao NAGANO ◽  
Ryo ICHINOHASAMA ◽  
Natsumi ASATO ◽  
Yoshiaki KONDO ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Growth Factor ◽  
Lysophosphatidic Acid ◽  
Culture Medium ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Platelet Derived Growth Factor ◽  
P53 Expression ◽  
Mesangial Cell Proliferation

Although mesangial cell death has been shown to be correlated with mesangial cell mitosis in vivo, little is known about how these two apparently opposite events are regulated. We show that the addition of platelet-derived growth factor (PDGF; 10–50 ng/ml) to primary cultured rat mesangial cells for 24 h caused continuous proliferation along with simultaneous cell death. This process was accompanied by the fragmentation of DNA into nucleosomal oligomers, the development of apoptotic morphological changes in the nucleus, and increased expression of p53. Accumulation of lactate dehydrogenase (LDH) was also observed in the culture medium, suggesting that both apoptosis and necrosis are involved in the cell death mechanisms observed. We also observed that addition of 30 µM lysophosphatidic acid (LPA) to the culture medium greatly suppressed PDGF-induced cell death, leading to synergistically enhanced mesangial cell proliferation. DNA fragmentation, p53 expression and LDH release were all suppressed by LPA. We suggest that PDGF is a bifunctional molecule in mesangial cells that evokes both cell proliferation and cell death simultaneously, whereas LPA is a survival factor. We speculate that PDGF and LPA may play important roles in the progression or exacerbation of proliferative glomerulonephritis.

scholarly journals Resveratrol attenuates high glucose-induced nucleus pulposus cell apoptosis and senescence through activating the ROS-mediated PI3K/Akt pathway

2018 ◽  
Vol 38 (2) ◽  
Author(s):  
Wenping Wang ◽  
Pei Li ◽  
Jiagang Xu ◽  
Xiangkun Wu ◽  
Zhiliang Guo ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
High Glucose ◽  
Cell Apoptosis ◽  
Culture Medium ◽  
Nucleus Pulposus Cell ◽  
Akt Pathway ◽  
Control Group ◽  
Ros Generation ◽  
Protective Effects ◽  
Potential Mechanism

Background: Diabetes mellitus is closely correlated with disc degeneration. Nucleus pulposus (NP) cell apoptosis and senescence are typical cellular features within the degenerative disc. Resveratrol is a newly identified phytoalexin that has protective effects on cartilaginous tissue. Objective: To investigate the whether resveratrol can protect against high glucose-induced NP cell apoptosis and senescence, and the potential mechanism in this process. Methods: Rat NP cells were cultured in either 10% FBS culture medium (control group) or 10% FBS with a high glucose concentration (0.2 M, experiment group) for 3 days. Resveratrol or the combination of resveratrol and LY294002 was added into the culture medium of experiment group to investigate the effects of resveratrol and the PI3K/Akt pathway. Results: High glucose significantly promoted NP cell apoptosis and NP cell senescence compared with the control group. Resveratrol exhibited protective effects against high glucose-induced NP cell apoptosis and senescence. Further analysis showed that resveratrol suppressed reactive oxygen species (ROS) generation and increased the activity of the PI3K/Akt pathway under the high glucose condition. However, the LY294002 had no significant effects on ROS content in the resveratrol-treated high glucose group. Conclusion: Resveratrol can attenuate high glucose-induced NP cell apoptosis and senescence, and the activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

scholarly journals The Expression and Significance of mTORC1 in Diabetic Retinopathy

2019 ◽  
Author(s):  
Yanli Liu ◽  
Yarong Zheng ◽  
Yekai Zhou ◽  
Yi Liu ◽  
Mengjuan Xie ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Diabetic Retinopathy ◽  
Western Blotting ◽  
Rat Model ◽  
High Glucose ◽  
Control Group ◽  
Diabetes Group ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background: To investigate the expression and significance of mechanistic target of rapamycin complex 1(mTORC1) in diabetic retinopathy(DR), and to find new targets and new methods for the treatment of DR.Methods: A DR rat model was prepared by general feeding combined with intraperitoneal injection of 10% streptozotocin (60 mg/kg). The rats were randomly divided into a control group (NDM group) and diabetes group (DM group).Three months later,the degrees of retinopathy were determined using hematoxylin and eosin staining,and the levels of p-S6, VEGF, and PEDF proteins were detected by immunohistochemistry and western blotting. Human retinal capillary endothelial cells (HRCECs) were cultured in high glucose conditions,then treated with rapamycin or transfected with siTSC1.The protein levels of p-S6 were assessed by western blotting. The 5-ethynyl-2´-deoxyuridine assay was used to detect cell proliferation, and the Transwell assay was used to detect cell migration.Results: A DM rat model was successfully developed. The expressions of p-S6 and VEGF proteins were significantly increased in the DM group (p < 0.05), and the expression of PEDF protein was significantly decreased compared with the control group (p < 0.05). In vitro,the p-S6 protein in high glucose(HG) induced HRCECs was increased compared with the normal control (p < 0.05), and cell proliferation and migration were increased compared with the normal glucose(NG) group (p < 0.05). After transfection with siTSC1 to activate mTORC1,the expression of p-S6 was increased,as well as cell proliferation and migration.In contrast rapamycin decreased p-S6 expression in HG induced HRCECs, as well as decrased proliferation and migration (p < 0.05).Conclusion: The mTORC1 played an important role in DR. After activation, mTORC1 induced expression of the p-S6 protein, regulated the expressions of VEGF and PEDF proteins, and changed the proliferation and migration of endothelial cells.The mTORC1 can therefore be used as a new target,as well as in the treatment of DR.

scholarly journals Extensive mechanical tension promotes annulus fibrosus cell senescence through suppressing cellular autophagy

2019 ◽  
Vol 39 (4) ◽  
Author(s):  
Liang Zhao ◽  
Baofang Tian ◽  
Qing Xu ◽  
Cunxin Zhang ◽  
Luo Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Disc Degeneration ◽  
Cell Biology ◽  
Annulus Fibrosus ◽  
Mechanical Load ◽  
Telomerase Activity ◽  
Cell Senescence ◽  
Mechanical Tension ◽  
Cellular Autophagy

Abstract Background: Mechanical load contributes a lot to the initiation and progression of disc degeneration. Annulus fibrosus (AF) cell biology under mechanical tension remains largely unclear. Objective: The present study was aimed to investigate AF cell senescence under mechanical tension and the potential role of autophagy. Methods: Rat AF cells were cultured and experienced different magnitudes (5% elongation and 20% elongation) of mechanical tension for 12 days. Control AF cells were kept static. Cell proliferation, telomerase activity, cell cycle fraction, and expression of senescence-related molecules (p16 and p53) and matrix macromolecules (aggrecan and collagen I) were analyzed to evaluate cell senescence. In addition, expression of Beclin-1 and LC3, and the ratio of LC3-II to LC3-I were analyzed to investigate cell autophagy. Results: Compared with the control group and 5% tension group, 20% tension group significantly decreased cell proliferation potency and telomerase activity, increased G1/G0 phase fraction, and up-regulated gene/protein expression of p16 and p53, whereas down-regulated gene/protein expression of aggrecan and collagen I. In addition, autophagy-related parameters such as gene/protein expression of Beclin-1 and LC3, and the ratio of LC3-II to LC3-I, were obviously suppressed in the 20% tension group. Conclusion: High mechanical tension promotes AF cell senescence though suppressing cellular autophagy. The present study will help us to better understand AF cell biology under mechanical tension and mechanical load-related disc degeneration.

scholarly journals Expression of miR-195 and its target gene Bcl-2 in human intervertebral disc degeneration and their effects on nucleus pulposus cell apoptosis

2020 ◽  
Author(s):  
Xue-Lin Lin ◽  
Zhao-Yun Zheng ◽  
Qing-Shan Zhang ◽  
Zhen Zhang ◽  
You-Zhi An
Keyword(s):  
Cell Proliferation ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Disc Degeneration ◽  
Western Blotting ◽  
Cell Apoptosis ◽  
Intervertebral Disc Degeneration ◽  
Target Gene ◽  
Blank Group ◽  
Tnf Α

Abstract Objective: To investigate the expression of miR-195 and its target gene Bcl-2 in intervertebral disc degeneration (IVDD) and its effect on nucleus pulposus (NP) cell apoptosis.Methods: The expressions of miR-195 and Bcl-2 in NP tissues of IVDD patients were quantified by qRT-PCR and Western blotting respectively. NP cells were divided into Blank group, TNF-α group, TNF-α + miR-NC group, TNF-α + siBcl-2 group, and TNF-α + miR-195 inhibitors + siBcl-2 group. Cell proliferation was detected by MTT assay, cell apoptosis evaluated by flow cytometry, mitochondrial membrane potential (MMP) tested by JC-1 staining, and the expression of apoptosis-related proteins quantified by Western blotting. Results: Compared with controls, IVDD patients had significantly increased miR-195 expression and decreased Bcl-2 protein in NP tissues. The expression of miR-195 was negatively correlated with the expression of Bcl-2 in NP tissues of IVDD patients (r = - 0.89, P < 0.001). Dual-luciferase reporter gene assay indicated that Bcl-2 was a target gene of miR-195. In comparison with Blank group, TNF-α group showed decreased cell proliferation and MMP, increased cell apoptosis, up-regulated expression of miR-195, Bax and cleaved caspase 3, and down-regulated Bcl-2 protein, these changes were attenuated by miR-195 inhibitors. Additionally, siBcl-2 can reverse the protective effect of miR-195 inhibitors on TNF-α-induced NP cells. Conclusion: IVDD patients had increased miR-195 expression in NP tissues, and inhibiting miR-195 can specifically up-regulate Bcl-2 expression to curb apoptosis of TNF-α-induced NP cells.

scholarly journals Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype

2020 ◽  
Author(s):  
Ako Yokoi ◽  
Toshihide Matsumoto ◽  
Yasuko Oguri ◽  
Yoshinori Hasegawa ◽  
Masataka Tochimoto ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Molecular Mechanisms ◽  
Biological Behavior ◽  
Clinical Samples ◽  
Cell Phenotype ◽  
Wild Type ◽  
Poor Prognostic Factor ◽  
P53 Expression ◽  
Mesenchymal Transition ◽  
Wild Type P53

Abstract Background We previously demonstrated that ovarian high grade serous carcinomas (OHGSeCa) and ovarian clear cell carcinomas (OCCCa) with an HNF-1β+/p53+/ARID1A+ immunophenotype were associated with the worst unfavorable prognosis. To clarify the molecular mechanisms underlying this finding, we focused on alterations in the p53 signaling pathway in these tumors. Methods Changes in cell phenotype and function following knockdown of wild-type p53 (p53-KD) were assessed using OCCCa cells expressing endogenous HNF-1β and ARID1A. The prognostic significance of molecules that were deregulated following p53-KD was also examined using 129 OCCCa/OHGSeCa cases. Results p53-KD cells had increased expression of Snail, phospho-Akt (pAkt), and pGSK3β, and decreased E-cadherin expression, leading to epithelial-mesenchymal transition (EMT)/cancer stem cell (CSC) features. The cells also exhibited acceleration of cell motility and inhibition of cell proliferation and apoptosis. Next generation sequencing assay revealed that fibronectin (FN) expression was significantly increased in the p53 KD-cells, in line with our observation that wild-type p53 (but not mutant p53) repressed FN1 promoter activity. In addition, treatment of OCCCa cells with FN significantly increased cell migration capacity and decreased cell proliferation rate, independent of induction of EMT features. In clinical samples, FN/p53 scores were significantly higher in OCCCa/OHGSeCa with the HNF-1β+/p53+/ARID1A+ immunophenotype when compared to others. Moreover, high FN/high p53 expression was associated with the worst overall survival and progression-free survival in OCCCa/OHGSeCa patients. Conclusion These findings suggest that upregulation of FN following loss of p53 function may impact the biological behavior of OCCCa/OHGSeCa, particularly in tumors with HNF-1β+/p53+/ARID1A+ immunophenotype, through alterations in cell mobility and cell proliferation. The accompanying induction of EMT/CSC properties and inhibition of apoptosis due to p53 abnormalities also contribute to the establishment and maintenance of tumor phenotypic characteristics.

scholarly journals miR-424 up-regulation inhibiting RF/6A cells function under high glucose condition via CCND1

2021 ◽  
Author(s):  
Yuan Chen ◽  
Hai-Ying Chen ◽  
Miao-Qin Wu ◽  
Zheng-Ru Huang
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Wound Healing ◽  
High Glucose ◽  
Vascular Dysfunction ◽  
Flow Cytometric Analysis ◽  
Tube Formation ◽  
Luciferase Reporter ◽  
Cell Phenotype ◽  
Scratch Wound

Abstract Backgrounds: Retinal vascular dysfunction is an important factor to the progression of diabetic retinopathy(DR). Multiple abnormal microRNAs (miRNAs) contributes to the pathogenesis of vascular dysfunction. However, the role and underlying mechanism of miR-424 in retinal vascular endothelial cells dysfunction under hyperglycemia stress remain obscure. Methods: Rhesus macaque choroid retinal endothelial cell line (RF/6A) cells were cultured under normal glucose (NG) and high glucose (HG) condition. qPCR was used to quantify the mRNA expression of miR-424 and Cyclin D1 (CCND1) and western blot was applied to detect the protein amount of CCND1. RF/6A cells were transfected with miR-424 mimics, miR-424 inhibitor, miR-424 inhibitor+ siRNA-CCND1 or with vehicle molecules. The cell proliferation, wound healing, tube formation ability and cell cycle were evaluated by MTT assay, scratch wound healing assay, tube formation assay and flow cytometric analysis, respectively. Interaction between miR-424 and CCND1 was predicted with bioinformatics and confirmed by Dual Luciferase Reporter Analysis.Results: Compared with NG, miR-424 was up-regulated and cell phenotype such as proliferation, wound healing and tube formation were inhibited in HG. The phenotypes can be reversed by inverting miR-424 expression under different conditions. CCND1 was confirmed as one of target genes of miR-424 and it can be modulated at transcriptional or translation level. Manipulation of silencing CCND1 can reverse the influences, such as promotion in proliferation, scratch wound tube formation and cell cycle, induced by transfecting miR-424 inhibitor into RF/6A cells under HG. Conclusions: Overexpression of miR-424 in RF/6A cells under HG stress significantly inhibited the cell function such as cell proliferation, wound healing, tube formation through suppressing CCND1 and blocking cell cycle.

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