MSK1 downregulation is involved in inflammatory responses following subarachnoid hemorrhage in rats

Abstract

Subarachnoid hemorrhage (SAH) is a life-threatening neurological disease. Recently, inflammatory factors have been confirmed to be responsible for the brain damage associated with SAH. Therefore, studying the post-SAH inflammatory reaction may clarify the mechanism of SAH. Mitogen and stress-activated protein kinase 1 (MSK1) causes the phosphorylation of NF-κB and regulates the activity of pro-inflammatory transcription factors. The present study aimed to identify the potential role of MSK1 in inflammation and brain damage development following SAH. A cisterna magna blood injection model was established in Sprague-Dawley rats. Hematoxylin and eosin staining, reverse transcription-quantitative polymerase chain reaction assays and double immunofluorescence staining were used to analyze the role of MSK1, IL-1β and TNF-α in the inflammatory process after SAH. In a model of lipopolysaccharide-induced astrocyte inflammation, the effect of overexpressing MSK1 overexpression was analyzed by western blot analysis. The results demonstrated that MSK1 expression were negatively correlated with TNF-α and IL-1β expression levels, and reached peak levels 2 days after TNF-α and IL-1β. The double immunofluorescence staining results showed that the expression of MSK1 was in the same plane of view as TNF-α and IL-1β in the brain cortex. Furthermore, the in vitro studies indicated that the overexpression of MSK1 inhibited the expression of TNF-α and IL-1β following LPS challenge. These results imply that MSK1 may be involved in the inflammatory reaction following SAH, and may potentially serve as a negative regulator of inflammation.

Keywords: inflammation; mitogen and stress-activated protein kinase 1; subarachnoid hemorrhage.

Figure 1

Animal model construction. Left image represents rat brain tissue from the sham group. The right image demonstrates rat brain tissue harvested 1 day after experimental subarachnoid hemorrhage.

Figure 2

Hematoxylin and eosin staining of inflammatory cells in cross sections in adult rat brain tissues. Local infiltration by inflammatory cells (predominantly monocytic and a neutrophilic granulocyte) increased markedly in the brain tissues at 1 and 3 days after subarachnoid hemorrhage compared with the sham group. Statistical evaluation of the three groups was performed with a one-way analysis of variance followed by Dunnett's post hoc test. ^*P<0.05 vs. sham group.

Figure 3

mRNA expression of MSK1, IL-1β, TNF-α in the brain cortex at various survival time intervals after SAH. The quantitative analysis demonstrated the high expression of MSK1 mRNA in the control and sham groups. The level of MSK1 decreased gradually after SAH, then peaked at 3 days, and was increased during the following days in the cortex. Conversely, analysis of the levels of IL-1β and TNF-α mRNA indicated that these mRNA gradually increased following SAH, reaching peak levels at 1 day, and then gradually decreased during the following days in the cortex. The correlation was determined by Spearman's analysis. Bars represent the mean ± SEM (n=6 in each group). Statistical analysis was performed using one-way analysis of variance followed by Dunnett's post-hoc test. ^*P<0.05 and ^**P<0.01 vs. sham and normal groups. MSK1, mitogen and stress-activated protein kinase 1; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; SAH, subarachnoid hemorrhage.

Figure 4

Double immunofluorescence staining for MSK1, TNF-α, IL-1β and GFAP in brain cortex tissues at 1 day after SAH. In adult rat brain tissues after injury, sections were labeled with MSK1 and GFAP, an astrocytes marker, and the co-localization of MSK1 with GFAP was demonstrated in the brain tissues. In addition, immunofluorescence staining for MSK1, TNF-α and IL-1β was also performed, and the merged images indicated that MSK1 was closely positioned to TNF-α and IL-1β, in the same view in each section. Scale bar=10 µm. MSK1, mitogen and stress-activated protein kinase 1; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; GFAP, glial fibrillary acidic protein.

Figure 5

Different expression levels of TNF-α induced by LPS in cultured primary astrocytes. Western blot analysis demonstrated that LPS induced astrocytes to express TNF-α in a dose-dependent manner. The bar chart indicates the ratio of TNF-α to β-actin. Statistical analysis of the groups was performed using a one-way analysis of variance followed by Dunnett's post-hoc test. ^*P<0.05 vs. sham group. TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide; N, negative control.

Figure 6

Western blot analysis of MSK1, IL-1β and TNF-α protein expression levels in cultured primary astrocytes post-transfection. MSK1 overexpression decreased the expression of TNF-α. β-actin was used as a control. Statistical analysis of the groups was performed using a one-way analysis of variance followed by Dunnett's post-hoc test. ^*P<0.05 and ^**P<0.01 vs. normal group. MSK1, mitogen and stress-activated protein kinase 1; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide; N, negative control.