The change tendency of endoplasmic reticulum stress associated proteins in rats with spinal cord injury

Abstract

To investigate endoplasmic reticulum (ER) stress reactions in spinal cord injury rats by evaluating the expression of the glucose-regulated protein 78 (GRP78), C/EBP homologous transcription factor protein (CHOP), X-box binding protein 1 (XBP1), Eif-2α and Bad. SCI models were established using adult female mice. After SCI, the expression of endoplasmic reticulum stress-induced apoptosis proteins were examined in the mice at specific time points using immunohistochemistry and western blot. The results of immunohistochemistry showed that in spinal cord gray matter, Chop, Grp78, XBP1, Eif-2α and Bad were specifically detected in the cytoplasm of the cell. Compare with the SCI group, there was little expression in normal group and sham group. The expression of ER stress-induced apoptosis proteins were significantly increased after spinal cord injury, and the absolute expression was higher than normal group (P < 0.05). Western-Blot results showed that compare with the SCI group, there were little expression of ER stress-induced apoptosis proteins in normal group and sham group. The expression of ER stress-induced apoptosis proteins were significantly increased after spinal cord injury, and the absolute expression was higher than normal group (P < 0.05). These results suggest that some ER stress-induced apoptosis proteins, such as Chop, Grp78, XBP1, Eif-2α and Bad, were activated after spinal cord injury, but the precise regulatory mechanisms remain unclear. In the future, understanding of the precise mechanism of ER stress-mediated apoptosis in SCI may lead to the development of novel treatment strategies.

Keywords: Chop; Eif-2α; Grp78; Spinal cord injury; endoplasmic reticulum stress-induced apoptosis.

Figure 1

Overexpression of CHOP after SCI. A. Spinal cord transected for different times were detected by immunostaining. Higher magnification view from the cells showed that CHOP expressed in cells with a remarkable neuronal phenotype in SCI group than the normal and sham group, and it was mainly expressed in the cytoplasm of the cell. Scale bar: 200 μm, 100 μm, 50 μm. B. Analysis of the positive cells of the immunohistochemistry results. *represents P < 0.05 versus normal group, **P < 0.01 versus normal group. Data are the mean values ± SEM, n = 4.

Figure 2

Overexpression of Grp78 after SCI. A. Spinal cord transected for different times were detected by immunostaining. Higher magnification view from the cells showed that Grp78 expressed in cells with a remarkable neuronal phenotype in SCI group than the normal and sham group, and it was mainly expressed in the cytoplasm of the cell. Scale bar: 200 μm, 100 μm, 50 μm. B. Analysis of the positive cells of the immunohistochemistry results. *represents P < 0.05 versus normal group, **P < 0.01 versus normal group. Data are the mean values ± SEM, n = 4.

Figure 3

Overexpression of XBP1 after SCI. A. Spinal cord transected for different times were detected by immunostaining. Higher magnification view from the cells showed that XBP1 expressed in cells with a remarkable neuronal phenotype in SCI group than the normal and sham group, and it was mainly expressed in the cytoplasm of the cell. Scale bar: 200 μm, 100 μm, 50 μm. B. Analysis of the positive cells of the immunohistochemistry results. *represents P < 0.05 versus normal group, **P < 0.01 versus normal group. Data are the mean values ± SEM, n = 4.

Figure 4

Overexpression of Eif-2α after SCI. A. Spinal cord transected for different times were detected by immunostaining. Higher magnification view from the cells showed that Eif-2α expressed in cells with a remarkable neuronal phenotype in SCI group than the normal and sham group, and it was mainly expressed in the cytoplasm of the cell. Scale bar: 200 μm, 100 μm, 50 μm. B. Analysis of the positive cells of the immunohistochemistry results. *represents P < 0.05 versus normal group, **P < 0.01 versus normal group. Data are the mean values ± SEM, n = 4.

Figure 5

Overexpression of Bad after SCI. A. Spinal cord transected for different times were detected by immunostaining. Higher magnification view from the cells showed that Bad expressed in cells with a remarkable neuronal phenotype in SCI group than the normal and sham group, and it was mainly expressed in the cytoplasm of the cell. Scale bar: 200 μm, 100 μm, 50 μm. B. Analysis of the positive cells of the immunohistochemistry results. *represents P < 0.05 versus normal group, **P < 0.01 versus normal group. Data are the mean values ± SEM, n = 4.

Figure 6

The expression of CHOP in different group by Western Blot. The expression of CHOP was significantly increased after spinal cord injury, and 1 day group was the highest expression, and the normal is the lowest. GAPDH was used as an internal loading control. Bar chart is the quantitation of relative expression of CHOP to GAPDH. *represents P < 0.05 versus the normal group, **P < 0.01 versus the normal group. Data are the mean values ± SEM, n = 4.

Figure 7

The expression of Grp78 in different group by Western Blot. The expression of Grp78 was significantly increased after spinal cord injury, and 7 day group was the highest expression, and the normal is the lowest. GAPDH was used as an internal loading control. Bar chart is the quantitation of relative expression of CHOP to GAPDH. *represents P < 0.05 versus the normal group, **P < 0.01 versus the normal group. Data are the mean values ± SEM, n = 4.

Figure 8

The expression of XBP1 in different group by Western Blot. The expression of XBP1 was significantly increased after spinal cord injury, and 1 day group was the highest expression, and the normal is the lowest. GAPDH was used as an internal loading control. Bar chart is the quantitation of relative expression of CHOP to GAPDH. *represents P < 0.05 versus the normal group, **P < 0.01 versus the normal group. Data are the mean values ± SEM, n = 4.

Figure 9

The expression of Eif-2α in different group by Western Blot. The expression of Eif-2α was significantly increased after spinal cord injury, and 1 day group was the highest expression, and the normal is the lowest. GAPDH was used as an internal loading control. Bar chart is the quantitation of relative expression of CHOP to GAPDH. *represents P < 0.05 versus the normal group, **P < 0.01 versus the normal group. Data are the mean values ± SEM, n = 4.

Figure 10

The expression of p-Eif-2α in different group by Western Blot. The expression of p-Eif-2α was significantly increased after spinal cord injury, and 7 day group was the highest expression, and the normal is the lowest. GAPDH was used as an internal loading control. Bar chart is the quantitation of relative expression of CHOP to GAPDH. *represents P < 0.05 versus the normal group, **P < 0.01 versus the normal group. Data are the mean values ± SEM, n = 4.

Figure 11

The expression of Bad in different group by Western Blot. The expression of Bad was significantly increased after spinal cord injury, and 7 day group was the highest expression, and the normal is the lowest. GAPDH was used as an internal loading control. Bar chart is the quantitation of relative expression of CHOP to GAPDH. *represents P < 0.05 versus the normal group, **P < 0.01 versus the normal group. Data are the mean values ± SEM, n = 4.

Figure 12

The expression of p-Bad in different group by Western Blot. The expression of p-Bad was significantly increased after spinal cord injury, and 3 day group was the highest expression, and the normal is the lowest. GAPDH was used as an internal loading control. Bar chart is the quantitation of relative expression of CHOP to GAPDH. *represents P < 0.05 versus the normal group, **P < 0.01 versus the normal group. Data are the mean values ± SEM, n = 4.