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, 15, 1744806919892100

Role of Bone Morphogenetic protein-2/4 in Astrocyte Activation in Neuropathic Pain

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Role of Bone Morphogenetic protein-2/4 in Astrocyte Activation in Neuropathic Pain

Lin Yang et al. Mol Pain.

Abstract

Background: Bone morphogenetic protein-2/4 (BMP2/4) has been recognized as promoters of astrocyte activity. Substantial evidence suggests that BMP2/4 may be elevated and plays a critical role in astrocyte activation upon spinal cord injury. Although neuropathic pain is similarly associated with astrocyte activation, the participation of BMP2/4 in this regard still remains unclear.

Methods: A rat model of neuropathic pain achieved by spinal nerve ligation at L5 was used to evaluate the expression of glial fibrillary acidic protein and BMP2/4 in the spinal cord in days 1, 4, 7, 10, and 14. Next, normal rats received intrathecal exogenous BMP2/4 and the antagonist Noggin to assess the effect of BMP2/4 on astrocyte activation. In both experiments, von Frey filaments were used to evaluate the changes in paw withdrawal threshold. In addition, Western blotting and immunofluorescence were performed to assess the expression of glial fibrillary acidic protein, BMP2/4, p-Smad 1/5/8, and phospho-signal transducer and activator of transcription-3 (p-STAT3) in the spinal cord.

Results: Firstly, spinal nerve ligation caused a significant increase in the expression of BMP4, while BMP2 levels remained unchanged. Secondly, exogenous BMP4 but not BMP2 induced a significant decrease in paw withdrawal threshold, along with the upregulation of glial fibrillary acidic protein. Moreover, exogenous BMP4 stimulated both p-Smad 1/5/8 and p-STAT3, while BMP2 only upregulated p-Smad 1/5/8. Finally, exogenous Noggin alleviated the decrease in paw withdrawal threshold induced by BMP4 and reduced astrocyte activation, as well as p-STAT3 upregulation.

Conclusions: Our results indicate only BMP4—and not BMP2—intervened in allodynia in rats by eliciting glial activation probably through both p-Smad 1/5/8 and p-STAT3 signaling.

Keywords: Neuropathic pain; bone morphogenetic protein; glial activation; signal transducer and activator of transcription 3.

Figures

Figure 1.
Figure 1.
SNL causes allodynia and GFAP upregulation in rats. (a) Each time point indicates mean ± standard error of the mean of PWT in five rats as measured using von Frey filaments. There was a significant effect on time, F(5, 40) = 85.62, p < 0.0001, or SNL, F(1, 8) = 954.6, p < 0.0001, and interaction, F(5, 40) = 67.07, p < 0.0001, on PWT observed 14 days after SNL. Compared with the Sham group in which PWT remained at the baseline of 15 g, there was a significant decrease in PWT in day 1 (SNL: 4.82±1.03 g), day 4 (SNL: 2.27±1.19 g), day 7 (SNL: 4.82 ± 1.03 g), day 10 (SNL: 2.07 ± 1.02 g), and day 14 (SNL: 2.20 ± 1.24 g) (p < 0.01). **p < 0.01 compared with the Sham group. (b and c) Representative results from Western blotting indicated the ratio between GFAP and GAPDH remained comparable in day 1 (p = 0.12), but significantly increased in the SNL group in days 4, 7, 10, and 14 (p < 0.01), compared with the Sham group. The SNL group had significantly increased GFAP levels from day 4 to day 14. n = 3 at each time point of both groups. **p < 0.01 compared with the Sham group. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; PWT: paw withdrawal threshold; SNL: spinal nerve ligation.
Figure 2.
Figure 2.
Expression and localization pattern of BMP2 and BMP4 after SNL. (a and b) Representative results from Western blotting, illustrating that SNL-induced BMP4 expression in the dorsal spinal cord was prominently increased in days 1, 4, 7, and 10 (p < 0.01), and day 14 (p < 0.05). In contrast, BMP2 expression in the SNL group decreased in day 4 (p < 0.05), yet remained comparable in day 1 (p = 0.27), day 7 (p = 0.23), day 10 (p = 0.33), and day 14 (p = 0.09). n = 3 at each time point for both groups. *p < 0.05 compared with the Sham group; **p < 0.01 compared with the Sham group. (c) Double immunofluorescence staining of GFAP (green) and BMP2 (red) or BMP4 (red) in the dorsal horn of spinal cord at day 7 after SNL; showing that both BMP2 and BMP4 were abundantly colocalized with GFAP. Scale bar = 50 µm. BMP: bone morphogenetic protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; SNL: spinal nerve ligation.
Figure 3.
Figure 3.
Intrathecal administration of BMP4 instead of BMP2-induced allodynia and glial activation. (a) Each time point indicates mean ±standard error of the mean of PWT in five rats measured with von Frey filaments, depicting the time course of the separate effects of exogenous BMP2 and BMP4. There was a significant effect on time, F(4.030, 48.36) = 6.324, p = 0.0003, left; F(7, 32) = 4.274, p = 0.002, right, or intrathecal injection, F(2, 12) = 47.09, p < 0.0001, left; F(1.735, 55.52) = 99.72, p < 0.0001, right, and interaction, F(14, 84) = 4.498, p < 0.0001, left; F(14, 64) = 3.001, p = 0.0014, right, on bilateral PMT observed seven days after intrathecal administration. Compared with the Sham group, rats in the BMP4 group showed a significant decrease in PWT from day 1 (left: 9.09 ± 1.49 g, p < 0.01; right: 6.61 ± 1.71 g, p < 0.05) to day 7 (left: 8.33 ± 1.67 g, p < 0.01; right: 5.36 ± 2.54 g, p < 0.01). In contrast, rats in the BMP2 group maintained their PWT at baseline over the following seven days (day 1 left: 13.63 ± 0.95 g, right: 13.18 ± 2.30 g; day 7: left: 14.09 ± 0.20 g, right: 12.23 ± 2.37 g, p > 0.05), which was comparable to the Sham group (except for the right PWT on day 6: 10.77 ± 1.67 g, p < 0.05). Significant statistical differences of PWT were also found between the BMP2 and BMP4 groups at each time point. *p < 0.05 compared with the Sham group; **p < 0.01 compared with the Sham group; #p < 0.05 compared with the BMP2 group; ##p < 0.01 compared with the BMP2 group. (b and c) Representative Western blotting indicating GFAP expression was increased in the BMP4 group in days 1, 4, and 7 compared with the Sham group (p < 0.05). However, no GFAP upregulation was detected in the BMP2 group compared with the Sham group (day 1: p = 0.51; day 4: p = 0.45; day 7: p = 0.18). Statistical differences in GFAP expression were also found between the BMP2 and BMP4 groups at each time point (p < 0.05). n = 3 at each time point for both groups. *p < 0.05 compared with the Sham group; #p < 0.05 compared with the BMP2 group. BMP: bone morphogenetic protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; PWT: paw withdrawal threshold; SNL: spinal nerve ligation.
Figure 4.
Figure 4.
Exogenous BMP4 but not BMP2 activated both p-Smad1/5/9 and p-STAT3 signaling. (a and b) Western blotting showed the expression of p-Smad1/5/9 potently increased in both the BMP4 and BMP2 groups in day 1 (BMP4: p < 0.01; BMP2: p < 0.05), day 4 (BMP4: p < 0.05; BMP2: p < 0.05), and day 7 (BMP4: p < 0.01; BMP2: p < 0.01) compared with the Sham group. On the other hand, p-Smad1/5/9 expressions between the BMP4 and BMP2 groups remained similar (P1: p = 0.1224; P4: p = 0.1826; P7: p = 0.6561). In contrast, p-STAT3 expression in the BMP4 group considerably increased in days 1, 4, and 7 compared with the Sham group (P1: p < 0.01; P4: p < 0.01; P7: p < 0.05) and the BMP2 group (P1: p < 0.01; P4: p < 0.01; P7: p < 0.05), while p-STAT3 expression in the BMP2 group remained comparable with the Sham group (P1: p = 0.5180; P4: p = 0.2867; P7: p = 0.0846). n = 3 at each time point for both groups. *p < 0.05 compared with the Sham group; **p < 0.01 compared with the Sham group; #p < 0.05 compared with the BMP2 group; ##p < 0.01 compared with the BMP2 group. (c) Immunofluorescence images performed one day after intrathecal administration showed the upregulated p-STAT3 in BMP4 group was prominently localized in the dorsal horn of the spinal cord, while there was hardly any expression of p-STAT3 in both the Sham and the BMP2 group. n = 2 at each time point for both groups. Scale bar = 50 µm. (d) Double-labeling immunofluorescence further showed these p-STAT3-positive cells (red) were almost completely accumulated in the nucleus (blue) of GFAP-positive astrocytes (green). n = 2 at each time point for both groups. Scale bar = 25 µm. BMP: bone morphogenetic protein; DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; STAT: signal transducer and activator of transcription.
Figure 5.
Figure 5.
Noggin inhibited rBMP4-induced allodynia, glial activation, and p-STAT3 upregulation in a dose-dependent manner. (a) Each time point indicates mean ± standard error of the mean of PWT in five rats measured with von Frey filaments in order to assess the antagonistic effect of Noggin on BMP4. There was a significant effect on time, F(7, 32) = 10.13, p < 0.0001, left; F(7, 32) = 6.492, p < 0.0001, right, or intrathecal injection, F(2.177, 69.65) = 52.25, p < 0.0001, left; F(1.869, 59.82) = 66.98, p < 0.0001, right, and interaction, F(21, 96) = 1.792, p = 0.0301, left; F(21, 96) = 2.329, p = 0.0029, right, on bilateral PMT observed seven days after intrathecal administration. Firstly, the PWT of rats in the NOG group was shown to be comparable with the Sham group from day 1 to day 7 after the intrathecal treatments (p > 0.05). Secondly, the PWT of the B + NL group decreased significantly compared with the Sham group from day 1 (left: 6.92 ± 2.13 g, right: 9.65 ± 1.99 g) to day 7 (left: 4.02 ± 1.68 g, right: 5.71 ± 1.27 g). In addition, there was a notable increase in bilateral PWT in the B + NH group in comparison with B + NL group from day 3 (B + NH group: left: 7.60 ± 1.36 g, right: 10.46 ± 2.73 g; B + NL group: left: 4.86 ± 1.80 g, right: 7.36 ± 1.65 g) and day 7 (B + NH group: left: 7.33 ± 1.67 g right: 9.90 ± 1.27 g; B + NL group: left: 4.02 ± 1.68 g, right: 5.71 ± 1.27 g). *p < 0.05 compared with the Sham group; **p < 0.01 compared with the Sham group; #p < 0.05 compared with the B + NL group; ##p < 0.01 compared with the B + NL group. (b and c) Western blotting was performed to evaluate the antagonistic effect of Noggin on BMP4-induced activation of GFAP and p-STAT3. The expression of GFAP and p-STAT3 were comparable between Sham and NOG groups (GFAP: day 1 p = 0.608, day 4 p = 0.740, day 7 p = 0.911; p-STAT3: day 1 p = 0.388, day 4 p = 0.175, day 7 p = 0.139). Furthermore, the GFAP and p-STAT3 expression in B + NL group significantly increased compared with the Sham group (p < 0.05). In addition, a notable decrease in GFAP and p-STAT3 expressions could be detected in B + NH group compared with B + NL group, suggesting rNoggin could dose-dependently relieve rBMP4-induced GFAP and STAT3 activation. n = 3 at each time point of all groups. *p < 0.05 compared with the Sham group; **p < 0.01 compared with the Sham group; #p < 0.05 compared with the B + NL group; ##p < 0.01 compared with the B + NL group. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; NH: ■; NL: ■; NOG: ■; PWT: paw withdrawal threshold; STAT: signal transducer and activator of transcription.

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