Exosomes from mesenchymal stem cells modulate endoplasmic reticulum stress to protect against nucleus pulposus cell death and ameliorate intervertebral disc degeneration in vivo

Abstract

Objectives: Intervertebral disc degeneration (IDD) is widely accepted as a cause of low back pain and related degenerative musculoskeletal disorders. Nucleus pulposus (NP) cell apoptosis which is related to excessive endoplasmic reticulum (ER) stress in the intervertebral disc (IVD) could aggravate IDD progression. Many studies have shown the therapeutic potential of exosomes derived from bone marrow mesenchymal stem cells (MSC-exos) in degenerative diseases. We hypothesized that the delivery of MSC-exos could modulate ER stress and inhibit excessive NP cell apoptosis during IDD. Methods: The ER stress levels were measured in normal or degenerative NP tissues for contrast. The effects of MSC-exos were testified in advanced glycation end products (AGEs) -induced ER stress in human NP cells. The mechanism involving AKT and ERK signaling pathways was investigated using RNA interference or signaling inhibitors. Histological or immunohistochemical analysis and TUNEL staining were used for evaluating MSC-exos therapeutic effects in vivo. Results: The ER stress level and apoptotic rate was elevated in degenerative IVD tissues. MSC-exos could attenuate ER stress-induced apoptosis by activating AKT and ERK signaling. Moreover, delivery of MSC-exos in vivo modulated ER stress-related apoptosis and retarded IDD progression in a rat tail model. Conclusions: These results highlight the therapeutic effects of exosomes in preventing IDD progression. Our work is the first to demonstrate that MSC-exos could modulate ER stress-induced apoptosis during AGEs-associated IVD degeneration.

Keywords: Mesenchymal stem cells; advanced glycation end products; endoplasmic reticulum stress; exosomes; intervertebral disc degeneration.

Figure 1

ER stress level during IDD in human NP tissues. (A) Representative histological images of normal or degenerative NP tissues in HE, Alcian blue and Masson staining. Magnification: 400 ×. Histological grades of NP tissues were evaluated and analyzed. *P < 0.05. (B-D) The protein levels of GRP78 and CHOP were analyzed by western blot analysis (B) and the relative quantitative data (C, D) was calculated accordingly. GAPDH was used as an internal control. *P < 0.05 vs NC group. (E) GRP78 mRNA level was measured by qRT-PCR in normal and degenerative NP tissues (left panel). A correlation between GRP78 mRNA level and Pfirrmann grades of NP tissues (n = 30) was determined by nonparametric linear regression (right panel). (F) CHOP mRNA level was analyzed by qRT-PCR (left panel) and linear regression confirmed a correlation between CHOP mRNA level and Pfirrmann grades of NP tissues (n = 30) (right panel). (G-H) Representative images of caspase-3 (G) and caspase-12 (H) expression was detected by immunofluorescence analysis and the relative fluorescence intensity was calculated in normal and degenerative NP tissues. Magnification: 400 ×. *P < 0.05 vs NC group.

Figure 2

Identification of human bone marrow MSC and exosomes (MSC-exos). (A) Representative images of MSC spindle-like morphology and adherence to plastic (scale bar: 50 μm). (B) The ability of MSC to differentiate into the osteogenic, chondrogenic, and adipogenic lineages was confirmed by Alizarin Red staining (left panel, scale bar: 50 μm), Oil Red O staining (middle panel, scale bar: 20 μm) and Alcian blue staining (right panel, scale bar: 50 μm), respectively. (C) Cell surface markers (CD90, CD105, CD73, CD34 and HLA-DR) of MSC was detected by flow cytometric analysis. (D) Particle size distribution of MSC-exos was measured by nanoparticle trafficking analysis (NTA). (E) A three-dimensional plot according to the NTA results showed the stereo picture of MSC-exos size distribution. (F) Typical image of MSC-exos morphology was captured by transmission electron microscopy (TEM) (Scale bar: 200 nm). (G) Protein markers of MSC-exos were detected by western blot analysis in exosomes and MSC cells. (H) Representative images of NP cells incubated with PBS or PKH26-labelled MSC-exos for 12 h. The nuclei of NP cells were stained by DAPI (blue). Magnification: 400 ×, scale bar: 20 μm.

Figure 3

MSC-exos reduced the GRP78 and GRP94 expression under the AGEs stimulation. The human NP cells were treated with AGEs (200 μg/mL in 24 h except for in the control group. MSC-exos-10, 50, 100 indicates that 10, 50 or 100 μg/mL exosomes were used in the corresponding groups. (A) The protein levels of GRP78 and GRP94 were measured by western blot analysis and the relative quantitative data was calculated accordingly. GAPDH was used as an internal control. (B) Representative images of GRP78 expression treated with different concentrations of MSC-exos under the stimulation of AGEs. The nuclei of NP cells were stained by DAPI. Magnification: 200 ×. (C) Representative images of GRP94 expression in different groups. The nuclei of NP cells were stained by DAPI. Magnification: 200 ×. (D) Quantitative analysis of fluorescence intensity using Image-Pro Plus 6.0 for GRP78 and GRP94 according to the immunofluorescence analysis results. Data were presented as the mean ± SD. *P < 0.05 vs. control group, ^#P < 0.05 vs. AGEs group.

Figure 4

MSC-exos attenuated the activation of caspase-3 and caspase-12 under the AGEs stimulation in human NP cells. The human NP cells were treated with AGEs (200 μg/mL) in 24 h except for in the control group. MSC-exos-10, 50, 100 indicates that 10, 50 or 100 μg/mL exosomes were used in the corresponding groups. (A) Representative western blotting assay and quantitative analysis of cleaved caspase-3 and cleaved caspase-12 level. GAPDH was used as an internal control. (B) Representative images of caspase-3 and caspase-12 expression treated with different concentrations of MSC-exos under the stimulation of AGEs. The nuclei of NP cells were stained by DAPI. Magnification: 200 ×. (C) Quantitative analysis of fluorescence intensity for caspase-3 and caspase-12 according to the immunofluorescence analysis results. (D) Representative images of TUNEL analysis in different group. The nuclei of NP cells were stained by DAPI. Magnification: 200 ×. (E) Quantitation of the ratio of apoptotic cells in total cells was measured according to the TUNEL staining. Data were presented as the mean ± SD. *P < 0.05 vs. control group, ^#P < 0.05 vs. AGEs group.

Figure 5

MSC-exos ameliorated the activation of UPR under the AGEs treatment. The MSC-exos group was cotreated with AGEs (200 μg/mL) and MSC-exos (100 μg/mL). (A) The protein expression levels of p-PERK, PERK, ATF6, p-IREα, IREα, XBP1, ATF4, and CHOP were measured by western blot analysis. GAPDH was used as an internal control. (B) Quantitative analysis of relative protein levels for p-PERK, ATF6, p-IREα, XBP1, ATF4, and CHOP. (C-E) The transcriptional levels of XBP1 (C), ATF4 (D) and CHOP (E) were analyzed by qRT-PCR. Data were presented as the mean ± SD. *P < 0.05 vs. control group, ^#P < 0.05 vs. AGEs group.

Figure 6

MSC-exos attenuated the AGEs-induced ER stress-induced apoptosis in human NP cells. The MSC-exos group was cotreated with AGEs (200 μg/mL) and MSC-exos (100 μg/mL). (A) There fragments of si-CHOP (173, 247 and 520) were synthesized and the silencing efficiency was assessed by the western blotting analysis. Data were presented as the mean ± SD. *P< 0.05 vs. control group. (B-E) Western blot analysis and the quantitative statistical analysis showed the protein levels of CHOP (C), cleaved caspase-12 (D) and cleaved caspase-3 (E) expression. GAPDH was used as an internal control. Data were presented as the mean ± SD. *P < 0.05 vs. control group, ^#P < 0.05 vs. AGEs group, ^$P < 0.05. (F) Representative images of caspase-3, caspase-12 and CHOP protein expression were detected by immunofluorescence staining. Magnification: 200 ×. (G-H) Representative images of TUNEL staining (G) and the quantitative statistical analysis (H) showed the rate of TUNEL-positive cells in different groups. Magnification: 200 ×. Data were presented as the mean ± SD. *P < 0.05 vs. control group, ^#P < 0.05 vs. AGEs group, ^$P < 0.05.

Figure 7

MSC-exos modulated the AGEs-induced ER stress through activating the AKT and ERK signaling in human NP cells. The MSC-exos group was cotreated with AGEs (200 μg/mL) and MSC-exos (100 μg/mL). (A) The protein levels of AKT, p-AKT, ERK and p-ERK were assessed by western blotting. LY294002 (LY) is a broad-spectrum inhibitor of PI3K/AKT. PD98059 (PD) could inhibit the phosphorylation of ERK1/2 efficiently. (B-C) Western blot analysis and the quantitative statistical analysis showed the protein levels of p-AKT (B) and p-ERK (C). (D-G) The protein levels of CHOP (E), cleaved caspase-12 (F), cleaved-caspase-3 (G) were measured by western blotting and analyzed statistically. GAPDH was used as an internal control. Data were presented as the mean ± SD. *P < 0.05 vs. control group, ^#P < 0.05 vs. AGEs group, ^$P < 0.05. (H) Representative images of caspase-3, caspase-12 and CHOP protein expression were assessed by immunofluorescence staining. Magnification: 200 ×. (I-J) Representative images of TUNEL staining (I) and the quantitative statistical analysis (J) showed the rate of TUNEL-positive cells in different groups. Magnification: 200 ×. Data were presented as the mean ± SD. *P < 0.05 vs. control group, ^#P < 0.05 vs. AGEs group, ^$P < 0.05.

Figure 8

MSC-exos ameliorated the ER stress-related apoptosis and retarded the IDD progression in vivo. (A) X-ray of rat tail with three independent discs at 0, 4, 8 weeks. White, blue and red arrows mean discs with PBS, AGEs or AGEs and MSC-exos injection respectively. (B) T2-weighted MRI of rat tail at 0, 4, 8 weeks. White, blue and red arrows mean discs with PBS, AGEs or AGEs and MSC-exos injection respectively. (C-D) Changes in DHI (%DHI) (C) based on the X-ray and Pfirrmann MRI grades (D) based on the MRI results in each group (n = 20 for each group). Data were presented as the mean ± SD. *P < 0.05. (E) Representative HE staining and immunohistochemical staining of GRP78, CHOP and caspase-3 expression in each group. Magnification: 25 × (scar bar = 500 μm) and 200 × (scar bar = 50 μm). (F) Histological grades were assessed according to the HE staining (n = 20 for each group). Data were presented as the mean ± SD. *P < 0.05. (G-H) Representative TUNEL staining images (G) of rat discs and the statistical analysis (H) of TUNEL-positive cells in each group. Data were presented as the mean ± SD. *P < 0.05.