Mesenchymal Stem Cells Protect Nucleus Pulposus Cells from Compression-Induced Apoptosis by Inhibiting the Mitochondrial Pathway

Abstract

Objective: Excessive apoptosis of nucleus pulposus cells (NPCs) induced by various stresses, including compression, contributes to the development of intervertebral disc degeneration (IVDD). Mesenchymal stem cells (MSCs) can benefit the regeneration of NPCs and delay IVDD, but the underlying molecular mechanism is poorly understood. This study aimed to evaluate the antiapoptosis effects of bone marrow-derived MSC (BMSC) on rat NPCs exposed to compression and investigate whether the mitochondrial pathway was involved.

Methods: BMSCs and NPCs were cocultured in the compression apparatus at 1.0 MPa for 36 h. Cell viability, apoptosis, mitochondrial function, and the expression of apoptosis-related proteins were evaluated.

Results: The results showed that coculturing with BMSCs increased the cell viability and reduced apoptosis of NPCs exposed to compression. Meanwhile, BMSCs could relieve the compression-induced mitochondrial damage of NPCs by decreasing reactive oxygen species level and maintaining mitochondrial membrane potential as well as mitochondrial integrity. Furthermore, coculturing with BMSCs suppressed the activated caspase-3 and activated caspase-9, decreased the expressions of cytosolic cytochrome c and Bax, and increased the expression of Bcl-2.

Conclusions: Our results suggest that BMSCs can protect against compression-induced apoptosis of NPCs by inhibiting the mitochondrial pathway and thus enhance our understanding on the MSC-based therapy for IVDD.

Figure 1

Cell viability of NPCs exposed to compression measuring by the CCK-8 assay. (a) NPCs were exposed to compression for 0, 12, 24, 36, or 48 h. (b) Coculture with BMSCs was applied to all-time point to verify the protective effects of BMSCs. NS means no statistical significant difference. NP cells without compression treatment as control. The data are expressed as mean ± SD from three independent experiments. (^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus control).

Figure 2

The antiapoptosis effect of BMSCs on NPCs exposed to compression. (a) The phase-contrast photomicrograph of NPCs. (b) Hoechst 33258 staining of NPCs. Apoptotic cells were characterized by the brightly stained condensed nuclei (indicated by arrows). (c) TUNEL staining of NPCs. (d) Representative images of cell apoptosis by flow cytometry analysis after Annexin V/PI dual staining. (e) Summary data showing the apoptosis rate in different groups. The cells at the early stage of apoptosis were stained with Annexin V+/PI−, and the cells at the late stage of apoptosis were stained with Annexin V+/PI+. CL means compression load. The data are expressed as mean ± SD from three independent experiments (^∗P < 0.05 and ^∗∗P < 0.01 versus control or NPC + BMSC + CL; ^#P < 0.05 and ^###P < 0.001 versus NPC + CL).

Figure 3

The effect of BMSCs on the compression-induced intracellular accumulation of ROS in NPCs. (a) Typical graphs of ROS imaged by fluorescence microscopy. (b) The intracellular ROS levels were measured by flow cytometry through DCFH-DA staining. (c) Summary data showing the mean fluorescence intensity (MFI) in different groups. CL means compression load. The data are expressed as mean ± SD from three independent experiments (^∗∗P < 0.01 and ^∗∗∗P < 0.001 versus control or NPC + BMSC + CL).

Figure 4

Coculture with BMSCs maintained the MMP of NPCs exposed to compression. (a) The MMP was analyzed by flow cytometry through JC-1 staining. (b) Summary data showing the quantitative MMP expressed as the ratio of red/green fluorescence intensity. (c) The representative fluorescence images of in situ JC-1 staining. CL means compression load. The data are expressed as mean ± SD from three independent experiments (^∗P < 0.05 and ^∗∗∗P < 0.001 versus control or NPC + BMSC + CL).

Figure 5

The mitochondrial ultrastructure of NPCs was assessed using TEM. (a) Control group and (b) NPC + BMSC group displayed normal mitochondria. (c) NPC + BMSC + CL group BMSCs improved the ultrastructure collapse of the mitochondria in NPCs with compression treatment. (d) NPC + CL group demonstrated disintegrating cristae and swelling mitochondria. CL means compression load (mitochondria were indicated by arrows).

Figure 6

The protein expression of cleaved caspase-3, Bax, Bcl-2, cleaved caspase-9, and cytosolic cytochrome c (cytosolic cyt c) determined by Western blotting. (a) The typical Western blot bands of cleaved caspase-3, Bax, Bcl-2, cleaved caspase-9, and cytosolic cytochrome c (cytosolic cyt c). (b), (c), (d), (e), (f) Summary data showing protein levels of cleaved caspase-3, Bax, Bcl-2, cleaved caspase-9, and cytosolic cytochrome c (cytosolic cyt c). CL means compression load. The data are expressed as mean ± SD from three independent experiments (^∗P < 0.05 and ^∗∗P < 0.01 versus control or NPC + BMSC + CL).