scholarly journals M2 Macrophage-Conditioned Medium Inhibits Intervertebral Disc Degeneration in a Tumor Necrosis Factor-α-rich Environment

2021 ◽  
Author(s):  
Xiao-Chuan Li ◽  
Shao-Jian Luo ◽  
Wu Fan ◽  
Tian-Li Zhou ◽  
Chun-Ming Huang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Intervertebral Disc ◽  
Disc Degeneration ◽  
Western Blotting ◽  
Intervertebral Disc Degeneration ◽  
M2 Macrophages ◽  
Matrix Synthesis ◽  
Culture Model ◽  
Tnf Α ◽  
Necrosis Factor

Abstract BackgroundInflammation is the primary pathological phenomenon associated with disc degeneration; accordingly, the inflammatory cytokine tumor necrosis factor (TNF-α) plays a crucial role in disc degeneration. M1 macrophages produce proinflammatory cytokines that facilitate the progression of intervertebral disc degeneration (IDD).However, the anti-inflammatory and regenerative effects of M2 macrophages on nucleus pulposus cells (NPCs) in IDD progression remain unknown. Here, we aimed to determine the role of M2 macrophages in IDD progression. MethodsM2 conditioned medium (M2CM) was harvested and purified from THP-1 cells; it was then used for culturing human NPCs and a mouse intervertebral disc (IVD) organ culture model. NPCs and IVD organ models were divided into the following three groups: group 1 was treated with 10% fetal bovine serum to actas the control, group 2 was treated with 10 ng/ml TNF-α, and group 3 was treated with 10 ng/ml TNF-α and M2CMto act as the co-culture group. After 3 to 14 days, cell proliferation (CCK-8 assay and western blotting for proliferation markers), extracellular matrix synthesis (quantitative polymerase chain reaction, western blotting, and immunofluorescence), apoptosis (TUNEL staining and western blotting), and NPC senescence (senescence-associated beta-galactosidase staining and western blotting) were assessed.ResultsCD206 and interleukin (IL)-10 levels were increased after 48 h of induction for M2 macrophages (both p<0.01). Cell proliferation was decreased in TNF-α-treated NPCs and was inhibited by M2CM co-culture. Moreover, TNF-α treatment enhanced the apoptosis, senescence, and expression of inflammatory factor-related genes, including IL-6, MMP-13, ADAMTS-4, and ADAMTS-5, whereas M2CM co-culture significantly reversed these effects. M2CM promoted aggrecan and collagen II synthesis but reduced collagen Iα1 levels in TNF-α treatment groups. Using our established three-dimensional murine IVD organ culture model, M2CM suppressed the inhibitory effect of TNF-α of the TNF-α-rich environment. ConclusionsCollectively, these results indicate that M2CM promotes cell proliferation and extracellular matrix synthesis and inhibits inflammation, apoptosis, and NPC senescence. This study therefore highlights the therapeutic potential of M2CM for IDD.

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

2021 ◽  
Vol 16 (1) ◽  
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 ◽  
Rat Model ◽  
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, and mitochondrial membrane potential (MMP) tested by JC-1 staining. Moreover, the function of miR-195 on IVDD in vivo was investigated using a puncture-induced IVDD rat model. Results IVDD patients had significantly increased miR-195 expression and decreased Bcl-2 protein expression in NP tissues. The expression of miR-195 was negatively correlated with the expression of Bcl-2 in IVDD patients. 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, upregulated expression of miR-195, Bax, and cleaved caspase 3, and downregulated Bcl-2 protein, while 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. Besides, inhibition of miR-195 alleviated IVDD degeneration and NP cell apoptosis in the rat model. Conclusion MiR-195 was significantly upregulated in NP tissues of IVDD patients, and inhibition of miR-195 could protect human NP cells from TNF-α-induced apoptosis via upregulation of Bcl-2.

scholarly journals JAG2/Notch2 inhibits intervertebral disc degeneration by modulating cell proliferation, apoptosis, and extracellular matrix

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Jun Long ◽  
Xiaobo Wang ◽  
Xianfa Du ◽  
Hehai Pan ◽  
Jianru Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Intervertebral Disc ◽  
Notch Signaling ◽  
Disc Degeneration ◽  
Intervertebral Disc Degeneration ◽  
Caspase 8 ◽  
Tnf Α ◽  
Intradiscal Injection

Abstract Background Intervertebral disc degeneration (IVDD)-related disorders are the major causes of low back pain. A previous study suggested that Notch activation serves as a protective mechanism and is a part of the compensatory response that maintains the necessary resident nucleus pulposus (NP) cell proliferation to replace lost or non-functional cells. However, the exact mechanism remains to be determined. In this study, we aimed to investigate the role of JAG2/Notch2 in NP cell proliferation and apoptosis. Methods Recombinant JAG2 or Notch2, Hes1, and Hey2 siRNAs were used to activate or inhibit Notch signaling. Cell proliferation, apoptosis, cell cycle regulatory factors, and pathways associated with Notch-mediated proliferation were examined. In vivo experiments involving an intradiscal injection of Sprague-Dawley rats were performed. Results Recombinant JAG2 induced Notch2 and Hes1/Hey2 expression together with NP cell proliferation. Downregulation of Notch2/Hes1/Hey2 induced G0/G1 phase cell cycle arrest in NP cells. Moreover, Notch2 mediated NP cell proliferation by regulating cyclin D1 and by activating PI3K/Akt and Wnt/β-catenin signaling. Furthermore, Notch signaling inhibited TNF-α-promoted NP cell apoptosis by suppressing the formation of the RIP1-FADD-caspase-8 complex. Finally, we found that intradiscal injection of JAG2 alleviated IVDD and that sh-Notch2 aggravated IVDD in a rat model. These results indicated that JAG2/Notch2 inhibited IVDD by modulating cell proliferation, apoptosis, and extracellular matrix. The JAG2/Notch2 axis regulated NP cell proliferation via PI3K/Akt and Wnt/β-catenin signaling and inhibited TNF-α-induced apoptosis by suppressing the formation of the RIP1-FADD-caspase-8 complex. Conclusions The current and previous results shed light on the therapeutic implications of targeting the JAG2/Notch2 axis to inhibit or reverse IVDD.

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

2021 ◽  
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 ◽  
Rat Model ◽  
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, and mitochondrial membrane potential (MMP) tested by JC-1 staining. Moreover, the function of miR-132 on IVDD in vivo was investigated using a puncture-induced IVDD rat model.Results: IVDD patients had significantly increased miR-195 expression and decreased Bcl-2 protein expression in NP tissues. The expression of miR-195 was negatively correlated with the expression of Bcl-2 in IVDD patients. 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, while 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. Besides, inhibition of miR-195 alleviated IVDD degeneration and NP cell apoptosis in the rat model.Conclusion: MiR-195 was significantly up-regulated in NP tissues of IVDD patients, and inhibition of miR-195 could protect human NP cells from TNF-α-induced apoptosis via upregulation of Bcl-2.

scholarly journals Serum CXCL12/SDF-1 level is positively related with lumbar intervertebral disc degeneration and clinical severity

2019 ◽  
Vol 26 (5) ◽  
pp. 341-350
Author(s):  
Zhao-Juan Er ◽  
Chun-Fang Yin ◽  
Wen-Jing Wang ◽  
Xue-Jun Chen
Keyword(s):  
Intervertebral Disc ◽  
Disc Degeneration ◽  
Roc Curve ◽  
Intervertebral Disc Degeneration ◽  
Curve Analysis ◽  
Clinical Severity ◽  
Roc Curve Analysis ◽  
Lumbar Intervertebral Disc ◽  
Tnf Α ◽  
Pfirrmann Grade

This study aimed to examine whether stromal cell-derived factor-1 (SDF-1) or C-X-C chemokine ligand 12 (CXCL12) participates in the development of lumbar disc degeneration, as implicated earlier by the level of CXCL12 correlating with this disease. It enrolled 145 patients with symptomatic lumbar intervertebral disc degeneration (IDD) and 130 asymptomatic healthy controls with no indication of IDD. Radiological assessment of the IDD patients was targeted at the lumbar vertebra region, based on Pfirrmann grade. Degeneration of the multifidus and psoas major muscles was evaluated using Goutallier classification. Visual Analogue Scale (VAS) and Oswestry Disability Index (ODI) scores were obtained for assessing the severity of manifestation. The levels of serum CXCL12, IL-6 and TNF-α were determined by ROC curve analysis, resulting in their prognostic value for Pfirrmann grading. Higher levels of serum CXCL12 were found in patients with IDD than in asymptomatic individuals, and were positively related to the Pfirrmann grade as well as multifidus muscle degeneration. Furthermore, serum CXCL12 concentration showed a significant correlation with the VAS and ODI scores. In addition, elevated serum CXCL12 levels were related to serum levels of TNF-α and IL-6. The ROC curve analysis implicated that CXCL12 could function as a biomarker of the early-mediate phase of IDD development. In summary, the serum CXCL12/SDF-1 level is positively related with lumbar IDD and its clinical severity.

Sign in / Sign up

Export Citation Format

Share Document