The role of IL-17 promotes spinal cord neuroinflammation via activation of the transcription factor STAT3 after spinal cord injury in the rat

Abstract

Study design: In this study, we investigated the role of IL-17 via activation of STAT3 in the pathophysiology of SCI.

Objective: The purpose of the experiments is to study the expression of IL-17 and related cytokines via STAT3 signaling pathways, which is caused by the acute inflammatory response following SCI in different periods via establishing an acute SCI model in rat.

Methods: Basso, Beattie, and Bresnahan hind limb locomotor rating scale was used to assess the rat hind limb motor function. Immunohistochemistry was used to determine the expression levels of IL-17 and p-STAT3 in spinal cord tissues. Western blotting analysis was used to determine the protein expression of p-STAT3 in spinal cord tissue. RT-PCR was used to analyze the mRNA expression of IL-17 and IL-23p19 in the spleen tissue. ELISA was used to determine the peripheral blood serum levels of IL-6, IL-21, and IL-23.

Results: Compared to the sham-operated group, the expression levels of IL-17, p-STAT3, IL-6, IL-21, and IL-23 were significantly increased and peaked at 24 h after SCI. The increased levels of cytokines were correlated with the SCI disease stages.

Conclusion: IL-17 may play an important role in promoting spinal cord neuroinflammation after SCI via activation of STAT3.

Figure 1

The severity of SCI was evaluated using hematoxylin-eosin staining (original magnification ×400). The spinal cord tissues were normal in the sham-operated group (a). The spinal cord demonstrated obvious sign of compression following SCI and the organization structure was destroyed as observed in (b). Next, the degree of inflammation gradually increased. A few scattered small foci of hemorrhage from small veins and capillaries and nerve cells are swollen and necrosis was observed at 24 h (c). The severity of the nerve cell necrosis and interstitial inflammatory cell infiltration were markedly increased at 48 h (d). Extensive hemorrhage and nerve cell necrosis was observed in at 72 h (e). Bar equals 50 μm.

Figure 2

(a) Representative IL-17 immunohistochemistry images in spinal cord tissue (brown granules, original magnification ×400). The sham-operated groups (A) and SCI group at 1 h (B), 24 h (C), 48 h (D), and 72 h (E). A digitized image shows labeled IL-17 immunohistochemistry in tissue. The gray value of the IL-17 immunohistochemical expression was assessed. IL-17 expression significantly increased in the rat SCI model group and peaked at 24 h. Bar equals 50 μm. (b) Morphometric quantitate of IL-17 protein expression in spinal cord tissues. The IOD in different groups was 0.19 ± 0.03, 0.24 ± 0.02, 0.40 ± 0.02, 0.33 ± 0.04, and 0.27 ± 0.04, respectively. Representative spinal cord tissue sections from the sham-operated group (n = 15) and SCI groups at 1 h (n = 15), 24 h (n = 15), 48 h (n = 15), and 72 h (n = 15) rats. The data are presented as the mean ± the SE. *P < 0.05 versus sham, ^# P < 0.05 versus 1 h, ^△ P < 0.05 versus 24 h, and ^● P < 0.05 versus 48 h.

Figure 3

(a) Representative p-STAT3 Immunohistochemical images in spinal cord tissue (brown granules, original magnification ×400). The sham-operated group (A) and SCI groups at 1 h (B), 24 h (C), 48 h (D), and 72 h (E). The digitized image shows labeled p-STAT3 immunohistochemistry on tissue. The IOD of p-STAT3 immunohistochemistry was assessed. The rat SCI model group showed significantly increased p-STAT3 expression and peaked at 24 h. Bar equals 50 μm. (b) Morphometric quantitate of p-STAT3 protein expression in spinal cord tissues. The IOD in different groups was 0.14 ± 0.02, 0.26 ± 0.02, 0.43 ± 0.03, 0.35 ± 0.02, and 0.30 ± 0.03, respectively. Representative spinal cord tissue sections from the sham-operated group (n = 15) and SCI groups at 1 h (n = 15), 24 h (n = 15), 48 h (n = 15), and 72 h (n = 15) rats. The data are presented as the mean ± the SE. *P < 0.05 versus sham, ^# P < 0.05 versus 1 h, ^△ P < 0.05 versus 24 h, and ^● P < 0.05 versus 48 h.

Figure 4

Phosphorylation of STAT3 protein levels in the spinal cord. The protein levels of p-STAT3 in the spinal cord tissues in different groups, as measured by western blotting analyses. The spinal cord tissues levels were 0.17 ± 0.04, 0.50 ± 0.02, 0.73 ± 0.03, 0.63 ± 0.04, and 0.37 ± 0.03, respectively, in the sham-operated group (n = 15) and SCI groups at 1 h (n = 15), 24 h (n = 15), 48 h (n = 15), and 72 h (n = 15) rats. The data are presented as the mean ± the SE. *P < 0.05 versus sham, ^# P < 0.05 versus 1 h, ^△ P < 0.05 versus 24 h, and ^● P < 0.05 versus 48 h.

Figure 5

(a) and (b): IL-17 and IL-23p19 mRNA transcription in spleen tissue. Representative images showed semiquantitative RT-PCR for IL-17 and IL-23p19 transcription sham. The sham-operated group; 1 h. The 1 h group of SCI; 24 h. The 24 h group of SCI; 48 h. The 48 h group of SCI; 72 h. The 72 h group of SCI. (c) and (d): densitometric quantification of IL-17 and IL-23p19mRNA expression in spleen tissues. Densitometric quantification of PCR bands showed that, among the IL-17 and IL-23p19, the sham-operated group, 1 h group of SCI, 24 h group of SCI, 48 h group of SCI, and 72 h group of SCI, the mRNA expression was significantly difference between each groups and peaked at 24 h. The data are presented as the mean ± the SE. *P < 0.05 versus sham, ^# P < 0.05 versus 1 h, ^△ P < 0.05 versus 24 h, and ^● P < 0.05 versus 48 h.

Figure 6

The IL-6, IL-21, and IL-23 levels of peripheral serum. The levels in the IL-6, IL-21, and IL-23 in the different groups, measured by ELISA. The IL-6 levels were 76.18 ± 2.36, 83.93 ± 1.85, 95.65 ± 3.97, 92.10 ± 2.64, and 88.81 ± 2.58. The IL-21 levels were 55.22 ± 5.59, 62.58 ± 3.91, 78.25 ± 6.82, 72.73 ± 5.18, and 67.65 ± 4.02. The IL-23 levels were 20.05 ± 2.07, 25.15 ± 1.51, 41.12 ± 3.19, 30.33 ± 1.69, and 22.42 ± 1.78, respectively, in the sham-operated group (n = 15), 1 h group of SCI (n = 15), 24 h group of SCI (n = 15), 48 h group of SCI (n = 15), and 72 h (n = 15) group of SCI. The data are presented as the mean ± the SE. *P < 0.05 versus sham, ^# P < 0.05 versus 1 h, ^△ P < 0.05 versus 24 h, and ^● P < 0.05 versus 48 h.