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Influence of simvastatin on the biological behavior of nucleus pulposus-derived mesenchymal stem cells.

ABSTRACT: Objectives:This research is to study the influences of different concentrations of simvastatin on the biological activities of nucleus pulposus-derived mesenchymal stem cells (NPMSC). Materials and Methods:NPMSC were cultured with different concentrations of simvastatin (0, 0.01, 0.1, and 1 ?M) and assessed to determine the possible effects of simvastatin. The cell proliferation was assessed with CCK-8 assay. The flowcytometry and multilineage differentiation were also performed to identify the stem characterization of the cells. The mRNA expressions of aggrecan, collagen type II, glucose transporter 1 (GLUT-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1? (HIF-1?) were determined by qRT-PCR. Results:The results demonstrated that the cells isolated from nucleus pulposus of healthy Sprague-Dawley (SD) rat met the criteria of MSC. NPMSC could form sunflower-like colonies and strongly expressed stem cell-related genes. In addition, NPMSC showed strong ability of chondrogenic, adipogenic and osteogenic differentiation. Simvastatin at certain range concentrations (0.01 ?M-0.1 ?M)) significantly promoted colony-forming rate and cell proliferation, and inhibited cell apoptosis. Simvastatin could promote expressions of aggrecan, collagen type II, HIF-1?, VEGF and GLUT-1, while 0.1 ?mol/l concentration reached the maximum effect. Our study further demonstrated that HIF-1?-intermediated signaling pathway might participate in regulating the biological activities of NPMSC. Conclusion:Proper concentration of simvastatin can promote the biological behavior of NPMSC, and HIF-1?-intermediated signaling pathway might participate in the mechanism.

SUBMITTER: Huang Z

PROVIDER: S-EPMC7043879 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Influence of simvastatin on the biological behavior of nucleus pulposus-derived mesenchymal stem cells.

Huang Zenan Z Cheng Xiaofei X Zhao Jie J Liu Zhongjun Z Wang Jingcheng J Feng Xinmin X Zhang Liang L

Iranian journal of basic medical sciences 20191201 12

<h4>Objectives</h4>This research is to study the influences of different concentrations of simvastatin on the biological activities of nucleus pulposus-derived mesenchymal stem cells (NPMSC).<h4>Materials and methods</h4>NPMSC were cultured with different concentrations of simvastatin (0, 0.01, 0.1, and 1 μM) and assessed to determine the possible effects of simvastatin. The cell proliferation was assessed with CCK-8 assay. The flowcytometry and multilineage differentiation were also performed t ...[more]

PMID: 32133066

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Project description:BACKGROUND:Stem cell therapy is considered as a promising alternative to treat intervertebral disc degeneration (IDD). Extensive work had been done on identifying and comparing different types of candidate stem cells, both in vivo and in vitro. However, few studies have shed light on degenerative nucleus pulposus cells (NPCs), especially their biological behavior under the influence of exogenous stem cells, specifically the gene expression and regulation pattern. In the present study, we aimed to determine messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs), which are differentially expressed during the co-culturing process with adipose-derived mesenchymal stem cells (ASCs) and to explore the involved signaling pathways and the regulatory networks. METHODS:We compared degenerative NPCs co-cultured with ASCs with those cultured solely using lncRNA-mRNA microarray analysis. Based on these data, we investigated the significantly regulated signaling pathways based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Moreover, 23 micro RNAs (miRNAs), which were demonstrated to be involved in IDD were chosen; we investigated their theoretic regulatory importance associated with our microarray data. RESULTS:We found 632 lncRNAs and 1682 mRNAs were differentially expressed out of a total of 40,716 probes. We then confirmed the microarray data by real-time PCR. Furthermore, we demonstrated 197 upregulated, and 373 downregulated Gene Ontology terms and 176 significantly enriched pathways, such as the mitogen-activated protein kinase (MAPK) pathway. Also, a signal-net was constructed to reveal the interplay among differentially expressed genes. Meanwhile, a mRNA-lncRNA co-expression network was constructed for the significantly changed mRNAs and lncRNAs. Also, the competing endogenous RNA (ceRNA) network was built. CONCLUSION:Our results present the first comprehensive identification of differentially expressed lncRNAs and mRNAs of degenerative NPCs, altered by co-culturing with ASCs, and outline the gene expression regulation pattern. These may provide valuable information for better understanding of stem cell therapy and potential candidate biomarkers for IDD treatment.

Project description:Intervertebral disc degeneration (IDD) is the primary pathological mechanism that underlies low back pain. Overloading-induced cell death, especially endogenous stem cell death, is the leading factor that undermines intrinsic repair and aggravates IDD. Previous research has separately studied the effect of oxygen concentration and mechanical loading in IDD. However, how these two factors synergistically influence endogenous repair remains unclear. Therefore, we established in vitro and in vivo models to study the mechanisms by which hypoxia interacted with overloading-induced cell death of the nucleus pulposus derived stem cells (NPSCs). We found the content of HIF1A (hypoxia inducible factor 1 subunit alpha) and the number of NPSCs decreased with disc degeneration in both rats and human discs. Hence, we isolated this subpopulation from rat discs and treated them simultaneously with hypoxia and excessive mechanical stress. Our results demonstrated that hypoxia exerted protective effect on NPSCs under compression, partially through elevating macroautophagy/autophagy. Proteomics and knockdown experiments further revealed HIF1A-BNIP3-ATG7 axis mediated the increase in autophagy flux, in which HMOX1 and SLC2A1 were also involved. Moreover, HIF1A-overexpressing NPSCs exhibited stronger resistance to over-loading induced apoptosis in vitro. They also showed higher survival rates, along with elevated autophagy after being intra-disc transplanted into over-loaded discs. Jointly, both in vivo and in vitro experiments proved the anti-apoptotic effect of HIF1A on NPSCs under the excessive mechanical loading, suggesting that restoring hypoxia and manipulating autophagy is crucial to maintain the intrinsic repair and to retard disc degeneration.Abbreviations: 3-MA: 3-methyladenine; ACAN: aggrecan; ATG7: autophagy related 7; BafA1: bafilomycin A1; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CASP3: caspase 3; CCK8: cell counting kit-8; CHT: chetomin; CMP: compression; CoCl2: cobalt chloride; COL2A1: collagen type II alpha 1 chain; Ctrl: control; DAPI: 4,6-diamidino-2-phenylindole; DEP: differentially expressed protein; DiR: 1,1-dioctadecyl-3,3,3,3-tetramethyl indotricarbocyanine; ECM: extracellular matrix; FCM: flow cytometry; GD2: disialoganglioside GD 2; GFP: green fluorescent protein; GO: gene ontology; GSEA: gene set enrichment analysis; H&E: hematoxylin-eosin; HIF1A: hypoxia inducible factor 1 subunit alpha; HK2: hexokinase 2; HMOX1: heme oxygenase 1; HX: hypoxia mimicry; IDD: intervertebral disc degeneration; IF: immunofluorescence; IHC: immunohistochemistry; IVD: intervertebral disc; KEGG: kyoto encyclopedia of genes and genomes; LBP: low back pain; Lv: lentivirus; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NC: negative control; NIR: near-infrared; NP: nucleus pulposus; NPC: nucleus pulposus cell; NPSC: nucleus pulposus derived stem cell; NX: normoxia; PPI: protein-protein interactions; RFP: red fluorescent protein; SLC2A1/GLUT1: solute carrier family 2 member 1; SQSTM1/p62: sequestosome 1; TEK/TIE2: TEK receptor tyrosine kinase; TEM: transmission electron microscopy; TUBB: tubulin beta class I.

Dataset Information

Influence of simvastatin on the biological behavior of nucleus pulposus-derived mesenchymal stem cells.

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Influence of simvastatin on the biological behavior of nucleus pulposus-derived mesenchymal stem cells.

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