N-cadherin Alleviates Apoptosis and Senescence of Nucleus Pulposus Cells via Suppressing ROS-dependent ERS in the Hyper-osmolarity Microenvironment

Figures

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  Figure 1. NP tissue from IDD patients exhibited a lower expression of NCDH and an enhanced occurrence rate of apoptosis and senescence. (A): Representative lumbar MRI photographs from LVF (Grade I, red arrows) and IDD (Grade IV, red arrows) patients were classified according to the Pfirrmann grading system. (B): HE staining of NP tissue from LVF and IDD patients. Scale bars: 50 μm. (C): Western blot analysis of protein expression of NCDH, P53, P21, P16, Cleaved-PARP1, Cleaved-caspase 3, Bcl-2, BIP and CHOP in NP tissue samples from LVF and IDD patients. (D): Immunofluorescence staining analysis of BIP expression in the NPCs from LVF and IDD patients. Scale bars: 50 μm. (E): TUNEL staining analysis of cell apoptosis of NP tissue from LVF and IDD patients. Scale bars: 50 μm. Note: The data represents mean ± SD (n=3); ***p<0.001, ****p<0.0001 vs. LVF group. LVF: lumbar vertebral fracture, IDD: intervertebral disc degeneration, NP: nucleus pulposus, NPCs: nucleus pulposus cells.

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  Figure 2. Hyper-osmolarity induces undesirable cell fates (apoptosis and senescence) and reduces NCDH expression of NPCs. (A): Western blot analysis of protein expression of NCDH, P53, P21, P16, Cleaved-PARP1, Bcl-2, Cleaved-Caspase-3, BIP and CHOP in NPCs in isotonicity (330 mOsm/kg) or hyper-osmolarity (550 mOsm/kg) culture. (B-C): Flow cytometry and SA-β-Gal staining of NPCs in isotonicity or hyper-osmolarity culture. Scale bars: 50 μm. (D): TEM analysis of ER morphology (red arrow) of NPCs in isotonicity or hyper-osmolarity culture. Scale bars: 1 μm. (E): DHE probe staining analysis of ROS content of NPCs in isotonicity or hyper-osmolarity culture. Scale bars: 50 μm. Note: The data represents mean ± SD (n=3); ***p<0.001, ****p<0.0001 vs. control. NP: nucleus pulposus, NPCs: nucleus pulposus cells, SA-β-Gal: Senescence-Associated β-Galactosidase, TEM: transmission electron microscopy, ROS: reactive oxygen species, DHE: dihydroethidium, ER: endoplasmic reticulum.

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  Figure 3. Hyper-osmolarity induces undesirable cell fates (apoptosis and senescence) of NPCs through ROS-dependent ERS. (A): Western blot analysis of expressions of NCDH, P53, P21, P16, Cleaved-PARP1, Cleaved-Caspase-3, Bcl-2, BIP and CHOP. (B): DHE staining analysis of ROS content. Scale bars: 50 μm. Note: The data represents mean ± SD (n=3); NS>0.05, ****p<0.0001 vs. hyper-osmolarity group. NPCs: nucleus pulposus cells, ROS: reactive oxygen species, DHE: dihydroethidium.

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  Figure 4. NCDH overexpression alleviates cellular undesirable cell fates (cell senescence and apoptosis) of NPCs in the hyper-osmolarity microenvironment. (A): Western bolt analysis of protein expression of NCDH, P53, P21, P16, Cleaved-PARP1, Cleaved-Caspase 3 and p-CDK1 (Thr-161) in NPCs after NCDH overexpression in the hyper-osmolarity culture. (B): SA-β-Gal staining analysis of cell senescence rate in NPCs. Scale bars: 50 μm. (C-D): Flow cytometry analysis of cell apoptosis and cell cycle in NPCs. Note: The data represents mean ± SD (n=3); **p<0.01, ***p<0.001, ****p<0.0001 vs. hyper-osmolarity with LV-NC group. NPCs: nucleus pulposus cells, SA-β-Gal: senescence-Associated β-Galactosidase.

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  Figure 5. P21 is the critical trigger to regulate apoptosis and senescence in NPCs under hyper-osmolarity microenvironment. (A): Western bolt analysis of protein expression of P21, BAD, Cleaved-Caspase-3, Cleaved-PARP1 and p-CDK1 (Thr-161) in NPCs with or without SiRNA treatment. (B): SA-β-Gal staining analysis of cell senescence rate in NPCs. Scale bars: 50 μm. (C-D): Flow cytometry analysis of cell apoptosis and cell cycle in NPCs. Note: The data represents mean ± SD (n=3); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. hyper-osmolarity with siRNA-P21 group. NPCs: nucleus pulposus cells, SA-β-Gal: senescence-Associated β-Galactosidase.

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  Figure 6. ATF4-CHOP axis is a key signaling transduction pathway behind the protective effects of NCDH overexpression against endoplasmic reticulum stress of NPCs in the hyper-osmolarity microenvironment. (A): Western blot analysis of protein expression of BIP, CHOP, ATF4, ATF6 and XPB1s in NPCs before and after NCDH overexpression in the hyper-osmolarity culture. (B): TEM analysis of ER morphology (red arrow) of NPCs after NCDH overexpression. Scale bars: 1 μm. (C): DHE probe staining analysis of ROS content in NPCs after NCDH overexpression. Scale bars: 50 μm. Note: The data represents mean ± SD (n=3); (D): Schematic diagram illustrates the protective effects of NCDH against a hyper-osmolarity microenvironment-induced undesirable cell fate of NPCs. ****p<0.0001 vs. hyper-osmolarity with LV-NC group. NPCs: nucleus pulposus cells, TEM: transmission electron microscopy, ER: endoplasmic reticulum, ROS: reactive oxygen species, DHE: dihydroethidium.

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  Figure 7. NCDH overexpression alleviates ERS and inhibits NP degeneration of rat tail disc in vivo. (A): T2-weighted MRI image of the experimental rat tail IVD. (B): HE staining of rat tail disc. Scale bars: 500 μm. (C): Western bolt analysis of protein expression of NCDH, Col2a1, Aggrecan and BIP. (D): Immunofluorescence staining of BIP expression. Scale bars: 50 μm. Note: The data represents mean ± SD (n=3); **p<0.01, ***p<0.001, ****p<0.0001 vs Puncture with LV-NC group.