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Jie Zhao
Shanghai Jiao Tong University School of Medicine
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1000-5641
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Lower back pain is often accredited to loss of intervertebral disc (IVD) height and compromised spine stability as a result of intervertebral disc degeneration (IVDD). We aim to locally use Bleomycin to induce the fibrotic transformation of bone marrow stromal cells (BMSCs) as a means to induce reparative fibrosis to slow down the height loss.
Methods: IVD from patients were gathered for histological examination. The expression of transforming growth factor beta 1 (TGF-β) signaling pathway was determined by qPCR and western blotting. Nucleus pulposus (NP) cells, annulus fibrosus (AF) cells and the Rats’ bone marrow stromal cells (BMSC)were cultured and their responsiveness to Bleomycin was evaluated by Cell Counting Kit-8, comet assay, transwell migration and wound healing assays. Rat IVDD models were created by puncture, rescued by Bleomycin injection and the effectiveness was evaluated by images (X-ray and MRI) and atomic force microscope.
Results: Histological examination showed increased levels of pro-fibrotic markers in IVDD tissues from patients. AF cells and BMSC cells was induced to adopt to a pro-fibrotic phenotype with increased expression fibrotic markers Col1a1, Col3a1, FSP1. The pro-fibrotic effect of Bleomycin on AF cells and BMSCs was in part due to the activation of the TGFβ-TGFβR1-SMAD2/3 signaling pathway. Pharmacological inhibition or gene knock-down of TGFβR1 could mitigate the pro-fibrotic effects.
Conclusion: Locally injection of Bleomycin in rats’ IVD induced rapid fibrosis and maintained its height through TGFβ-TGFβR1-SMAD2/3 signaling pathway.
Keywords
Intervertebral Disc Degeneration, Bleomycin, Bone Marrow Stromal Cells, Annulus Fibrosus Cells, Reparative Fibrosis, TGFβ-TGFβR1-SMAD2/3 Signaling Pathway
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This is a list of supplementary files associated with this preprint. Click to download.
- SUPFigure1.tif
(a) BMSCs were treated with Bleomycin in a concentration of 0, 5μg/ml and stained with Annexin V and PI, then subjected to flow cytometric analysis. (b) Quantification of early and late apoptotic cells rate using Graphpad8.0 by ordinary one-way ANOVA test. (c) BMSCs were treated with Bleomycin in a concentration of 0, 5μg/ml then stained with Comet assay kit. (g) The degree of DNA damage was measured by comet tail distance using Graphpad8.0 by ordinary one-way ANOVA test. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure1.tif
(a) BMSCs were treated with Bleomycin in a concentration of 0, 5μg/ml and stained with Annexin V and PI, then subjected to flow cytometric analysis. (b) Quantification of early and late apoptotic cells rate using Graphpad8.0 by ordinary one-way ANOVA test. (c) BMSCs were treated with Bleomycin in a concentration of 0, 5μg/ml then stained with Comet assay kit. (g) The degree of DNA damage was measured by comet tail distance using Graphpad8.0 by ordinary one-way ANOVA test. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure2.tif
(a) Cell death rate calculated by Graphpad8.0 by ordinary one-way ANOVA test of cells described at Figure 2d. (b) Western blot analysis of CyclinD1 and Cleaved Caspase3 in AF and NP cells stimulated with bleomycin of 0, 5 and 10μg/ml. (c) Q-PCR analysis of the relative mRNA expression levels of TGFβ, TGFβR1, Col1a1 and Fn1 in NP cells with Bleomycin.(d) Western blot analysis of phospho-Smad2, phospho-Smad3, Smad2 and Smad3 in NP cells with Bleomycin. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure2.tif
(a) Cell death rate calculated by Graphpad8.0 by ordinary one-way ANOVA test of cells described at Figure 2d. (b) Western blot analysis of CyclinD1 and Cleaved Caspase3 in AF and NP cells stimulated with bleomycin of 0, 5 and 10μg/ml. (c) Q-PCR analysis of the relative mRNA expression levels of TGFβ, TGFβR1, Col1a1 and Fn1 in NP cells with Bleomycin.(d) Western blot analysis of phospho-Smad2, phospho-Smad3, Smad2 and Smad3 in NP cells with Bleomycin. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure3.tif
(a, b, c, d) Quantification of distance in wound healing assay and migration rate of transwell test for cells described at figure 4a, b, c, d.(e) Q-PCR analysis of the relative mRNA expression levels of MMP3, MMP13 and Timp1 in NP cells with Bleomycin and Ly363937 or not. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure3.tif
(a, b, c, d) Quantification of distance in wound healing assay and migration rate of transwell test for cells described at figure 4a, b, c, d.(e) Q-PCR analysis of the relative mRNA expression levels of MMP3, MMP13 and Timp1 in NP cells with Bleomycin and Ly363937 or not. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- supFigure4.tif
(a) NP cells and BMSCs were treated with Bleomycin in a concentration of 5μg/ml once or three times and stained with β-gal staining buffer. (b) Quantification of the cells percentage stained with β-gal or not of cells in Sup figure 4a. (c) NP cells and BMSCs were treated with Bleomycin in a concentration of 5μg/ml once stained with PI buffer with RNase A, then subjected to flow cytometric analysis. (d) Quantification of the cells distribution by Cell Cycles Simulation in Sup figure 4c. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- supFigure4.tif
(a) NP cells and BMSCs were treated with Bleomycin in a concentration of 5μg/ml once or three times and stained with β-gal staining buffer. (b) Quantification of the cells percentage stained with β-gal or not of cells in Sup figure 4a. (c) NP cells and BMSCs were treated with Bleomycin in a concentration of 5μg/ml once stained with PI buffer with RNase A, then subjected to flow cytometric analysis. (d) Quantification of the cells distribution by Cell Cycles Simulation in Sup figure 4c. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure5.tif
(a) Q-PCR analysis of the relative mRNA expression levels of CDK1, CDK2, CDK4, CDK6, CCND2, CCNG2 and P21 in BMSCs with Bleomycin or not. (b) Western blot analysis of the protein expression levels of PARP, cleaved PARP, P21 and P53 in BMSCs with Bleomycin or not. (d) Immunofluorescence assay of Col2a1 in the fibrosis NP region and AF region described in Figure6c.
- SUPFigure5.tif
(a) Q-PCR analysis of the relative mRNA expression levels of CDK1, CDK2, CDK4, CDK6, CCND2, CCNG2 and P21 in BMSCs with Bleomycin or not. (b) Western blot analysis of the protein expression levels of PARP, cleaved PARP, P21 and P53 in BMSCs with Bleomycin or not. (d) Immunofluorescence assay of Col2a1 in the fibrosis NP region and AF region described in Figure6c.
- SUPFigure6.jpg
All rats were punctured at Co7/8, and tails of operation (Co6/7, Co7/8) were dissected and used to make paraffin section. (a,b) Q-PCR analysis of the relative mRNA expression levels of TGFβR2, TGFβR3 and Col3a1 in AF cells with Bleomycin or/and LY364947, with or without TGFβR1 knocked-down stimulated by Bleomycin. (c) Immunofluorescence assay of TGFβ, TGFβR1, FSP1 and Col1a1 in the fibrosis NP region and AF region. (d) IOD level of the red region described in a and Figure 6e were analyzed by IPP.6.0 and subsequently calculated with Graphpad8.0 by ordinary one-way ANOVA. (e) Safranin O-Fast Green stain and Sirius Red stain of the paraffin section. (f) Proportion quantification of area represent AF region, fibrosis NP region in Safranin O-Fast Green stain, Col1a1, Col3a1 and fibrosis NP region in Sirius Red stain using IPP6.0 and calculated by Graphpad8.0 by Student-t test. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001.
- SUPFigure6.jpg
All rats were punctured at Co7/8, and tails of operation (Co6/7, Co7/8) were dissected and used to make paraffin section. (a,b) Q-PCR analysis of the relative mRNA expression levels of TGFβR2, TGFβR3 and Col3a1 in AF cells with Bleomycin or/and LY364947, with or without TGFβR1 knocked-down stimulated by Bleomycin. (c) Immunofluorescence assay of TGFβ, TGFβR1, FSP1 and Col1a1 in the fibrosis NP region and AF region. (d) IOD level of the red region described in a and Figure 6e were analyzed by IPP.6.0 and subsequently calculated with Graphpad8.0 by ordinary one-way ANOVA. (e) Safranin O-Fast Green stain and Sirius Red stain of the paraffin section. (f) Proportion quantification of area represent AF region, fibrosis NP region in Safranin O-Fast Green stain, Col1a1, Col3a1 and fibrosis NP region in Sirius Red stain using IPP6.0 and calculated by Graphpad8.0 by Student-t test. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001.
- SUPFigure7.tif
(a, c) Immunofluorescence assay of KRT18 and S100A4 in the fibrosis NP region and AF region. (b, d) The ratio IOD level between the gree region and the red region described in Sup Figure 7a were analyzed by IPP.6.0 and subsequently calculated with Graphpad8.0 by ordinary one-way ANOVA. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure7.tif
(a, c) Immunofluorescence assay of KRT18 and S100A4 in the fibrosis NP region and AF region. (b, d) The ratio IOD level between the gree region and the red region described in Sup Figure 7a were analyzed by IPP.6.0 and subsequently calculated with Graphpad8.0 by ordinary one-way ANOVA. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- supFIGURE8.jpg
(a, c) Atomic Force Microscopic of fibrosis NP region in the paraffin section mentioned in Figure6 and Quantification of Young’s Modulus. (b, d) Evaluation of Topography-Displacement, Adhesion Force-Displacement, Young’s Modulus-Displacement and Deformation-Displacement curve. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- supFIGURE8.jpg
(a, c) Atomic Force Microscopic of fibrosis NP region in the paraffin section mentioned in Figure6 and Quantification of Young’s Modulus. (b, d) Evaluation of Topography-Displacement, Adhesion Force-Displacement, Young’s Modulus-Displacement and Deformation-Displacement curve. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
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Jie Zhao
Shanghai Jiao Tong University School of Medicine
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1000-5641
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View the published article at Stem Cell Research & Therapy.
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Background: Lower back pain is often accredited to loss of intervertebral disc (IVD) height and compromised spine stability as a result of intervertebral disc degeneration (IVDD). We aim to locally use Bleomycin to induce the fibrotic transformation of bone marrow stromal cells (BMSCs) as a means to induce reparative fibrosis to slow down the height loss.
Methods: IVD from patients were gathered for histological examination. The expression of transforming growth factor beta 1 (TGF-β) signaling pathway was determined by qPCR and western blotting. Nucleus pulposus (NP) cells, annulus fibrosus (AF) cells and the Rats’ bone marrow stromal cells (BMSC)were cultured and their responsiveness to Bleomycin was evaluated by Cell Counting Kit-8, comet assay, transwell migration and wound healing assays. Rat IVDD models were created by puncture, rescued by Bleomycin injection and the effectiveness was evaluated by images (X-ray and MRI) and atomic force microscope.
Results: Histological examination showed increased levels of pro-fibrotic markers in IVDD tissues from patients. AF cells and BMSC cells was induced to adopt to a pro-fibrotic phenotype with increased expression fibrotic markers Col1a1, Col3a1, FSP1. The pro-fibrotic effect of Bleomycin on AF cells and BMSCs was in part due to the activation of the TGFβ-TGFβR1-SMAD2/3 signaling pathway. Pharmacological inhibition or gene knock-down of TGFβR1 could mitigate the pro-fibrotic effects.
Conclusion: Locally injection of Bleomycin in rats’ IVD induced rapid fibrosis and maintained its height through TGFβ-TGFβR1-SMAD2/3 signaling pathway.
Figure 1
Figure 1
Figure 2
Figure 2
Figure 3
Figure 3
Figure 4
Figure 4
Figure 5
Figure 5
Figure 6
Figure 6
Figure 7
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- SUPFigure1.tif
(a) BMSCs were treated with Bleomycin in a concentration of 0, 5μg/ml and stained with Annexin V and PI, then subjected to flow cytometric analysis. (b) Quantification of early and late apoptotic cells rate using Graphpad8.0 by ordinary one-way ANOVA test. (c) BMSCs were treated with Bleomycin in a concentration of 0, 5μg/ml then stained with Comet assay kit. (g) The degree of DNA damage was measured by comet tail distance using Graphpad8.0 by ordinary one-way ANOVA test. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure1.tif
(a) BMSCs were treated with Bleomycin in a concentration of 0, 5μg/ml and stained with Annexin V and PI, then subjected to flow cytometric analysis. (b) Quantification of early and late apoptotic cells rate using Graphpad8.0 by ordinary one-way ANOVA test. (c) BMSCs were treated with Bleomycin in a concentration of 0, 5μg/ml then stained with Comet assay kit. (g) The degree of DNA damage was measured by comet tail distance using Graphpad8.0 by ordinary one-way ANOVA test. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure2.tif
(a) Cell death rate calculated by Graphpad8.0 by ordinary one-way ANOVA test of cells described at Figure 2d. (b) Western blot analysis of CyclinD1 and Cleaved Caspase3 in AF and NP cells stimulated with bleomycin of 0, 5 and 10μg/ml. (c) Q-PCR analysis of the relative mRNA expression levels of TGFβ, TGFβR1, Col1a1 and Fn1 in NP cells with Bleomycin.(d) Western blot analysis of phospho-Smad2, phospho-Smad3, Smad2 and Smad3 in NP cells with Bleomycin. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure2.tif
(a) Cell death rate calculated by Graphpad8.0 by ordinary one-way ANOVA test of cells described at Figure 2d. (b) Western blot analysis of CyclinD1 and Cleaved Caspase3 in AF and NP cells stimulated with bleomycin of 0, 5 and 10μg/ml. (c) Q-PCR analysis of the relative mRNA expression levels of TGFβ, TGFβR1, Col1a1 and Fn1 in NP cells with Bleomycin.(d) Western blot analysis of phospho-Smad2, phospho-Smad3, Smad2 and Smad3 in NP cells with Bleomycin. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure3.tif
(a, b, c, d) Quantification of distance in wound healing assay and migration rate of transwell test for cells described at figure 4a, b, c, d.(e) Q-PCR analysis of the relative mRNA expression levels of MMP3, MMP13 and Timp1 in NP cells with Bleomycin and Ly363937 or not. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure3.tif
(a, b, c, d) Quantification of distance in wound healing assay and migration rate of transwell test for cells described at figure 4a, b, c, d.(e) Q-PCR analysis of the relative mRNA expression levels of MMP3, MMP13 and Timp1 in NP cells with Bleomycin and Ly363937 or not. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- supFigure4.tif
(a) NP cells and BMSCs were treated with Bleomycin in a concentration of 5μg/ml once or three times and stained with β-gal staining buffer. (b) Quantification of the cells percentage stained with β-gal or not of cells in Sup figure 4a. (c) NP cells and BMSCs were treated with Bleomycin in a concentration of 5μg/ml once stained with PI buffer with RNase A, then subjected to flow cytometric analysis. (d) Quantification of the cells distribution by Cell Cycles Simulation in Sup figure 4c. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- supFigure4.tif
(a) NP cells and BMSCs were treated with Bleomycin in a concentration of 5μg/ml once or three times and stained with β-gal staining buffer. (b) Quantification of the cells percentage stained with β-gal or not of cells in Sup figure 4a. (c) NP cells and BMSCs were treated with Bleomycin in a concentration of 5μg/ml once stained with PI buffer with RNase A, then subjected to flow cytometric analysis. (d) Quantification of the cells distribution by Cell Cycles Simulation in Sup figure 4c. All data are presented as mean ±s. d. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure5.tif
(a) Q-PCR analysis of the relative mRNA expression levels of CDK1, CDK2, CDK4, CDK6, CCND2, CCNG2 and P21 in BMSCs with Bleomycin or not. (b) Western blot analysis of the protein expression levels of PARP, cleaved PARP, P21 and P53 in BMSCs with Bleomycin or not. (d) Immunofluorescence assay of Col2a1 in the fibrosis NP region and AF region described in Figure6c.
- SUPFigure5.tif
(a) Q-PCR analysis of the relative mRNA expression levels of CDK1, CDK2, CDK4, CDK6, CCND2, CCNG2 and P21 in BMSCs with Bleomycin or not. (b) Western blot analysis of the protein expression levels of PARP, cleaved PARP, P21 and P53 in BMSCs with Bleomycin or not. (d) Immunofluorescence assay of Col2a1 in the fibrosis NP region and AF region described in Figure6c.
- SUPFigure6.jpg
All rats were punctured at Co7/8, and tails of operation (Co6/7, Co7/8) were dissected and used to make paraffin section. (a,b) Q-PCR analysis of the relative mRNA expression levels of TGFβR2, TGFβR3 and Col3a1 in AF cells with Bleomycin or/and LY364947, with or without TGFβR1 knocked-down stimulated by Bleomycin. (c) Immunofluorescence assay of TGFβ, TGFβR1, FSP1 and Col1a1 in the fibrosis NP region and AF region. (d) IOD level of the red region described in a and Figure 6e were analyzed by IPP.6.0 and subsequently calculated with Graphpad8.0 by ordinary one-way ANOVA. (e) Safranin O-Fast Green stain and Sirius Red stain of the paraffin section. (f) Proportion quantification of area represent AF region, fibrosis NP region in Safranin O-Fast Green stain, Col1a1, Col3a1 and fibrosis NP region in Sirius Red stain using IPP6.0 and calculated by Graphpad8.0 by Student-t test. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001.
- SUPFigure6.jpg
All rats were punctured at Co7/8, and tails of operation (Co6/7, Co7/8) were dissected and used to make paraffin section. (a,b) Q-PCR analysis of the relative mRNA expression levels of TGFβR2, TGFβR3 and Col3a1 in AF cells with Bleomycin or/and LY364947, with or without TGFβR1 knocked-down stimulated by Bleomycin. (c) Immunofluorescence assay of TGFβ, TGFβR1, FSP1 and Col1a1 in the fibrosis NP region and AF region. (d) IOD level of the red region described in a and Figure 6e were analyzed by IPP.6.0 and subsequently calculated with Graphpad8.0 by ordinary one-way ANOVA. (e) Safranin O-Fast Green stain and Sirius Red stain of the paraffin section. (f) Proportion quantification of area represent AF region, fibrosis NP region in Safranin O-Fast Green stain, Col1a1, Col3a1 and fibrosis NP region in Sirius Red stain using IPP6.0 and calculated by Graphpad8.0 by Student-t test. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001.
- SUPFigure7.tif
(a, c) Immunofluorescence assay of KRT18 and S100A4 in the fibrosis NP region and AF region. (b, d) The ratio IOD level between the gree region and the red region described in Sup Figure 7a were analyzed by IPP.6.0 and subsequently calculated with Graphpad8.0 by ordinary one-way ANOVA. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- SUPFigure7.tif
(a, c) Immunofluorescence assay of KRT18 and S100A4 in the fibrosis NP region and AF region. (b, d) The ratio IOD level between the gree region and the red region described in Sup Figure 7a were analyzed by IPP.6.0 and subsequently calculated with Graphpad8.0 by ordinary one-way ANOVA. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- supFIGURE8.jpg
(a, c) Atomic Force Microscopic of fibrosis NP region in the paraffin section mentioned in Figure6 and Quantification of Young’s Modulus. (b, d) Evaluation of Topography-Displacement, Adhesion Force-Displacement, Young’s Modulus-Displacement and Deformation-Displacement curve. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
- supFIGURE8.jpg
(a, c) Atomic Force Microscopic of fibrosis NP region in the paraffin section mentioned in Figure6 and Quantification of Young’s Modulus. (b, d) Evaluation of Topography-Displacement, Adhesion Force-Displacement, Young’s Modulus-Displacement and Deformation-Displacement curve. All data are presented as mean ±sd. from three experiments. *P＜0.05, **P＜0.01， ***P＜0.001 and ****P＜0.0001
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