miR-221 activates Sox11 to reduce brain injury after intracerebral hemorrhage via inhibiting neuroinflammation

Abstract

As a global public health issue, intracerebral hemorrhage (ICH) is characterized by high morbidity and mortality. Brain injury following ICH is composed of primary and secondary injury, with the latter being more severe and resulting in increased apoptosis. Sox11 (sex-determining region Y-related high-mobility-group 11), a vital member of the Sox gene family, is broadly discovered in the developing nervous system and may have a vital impact on neurogenesis, neuronal survival, and neurite outgrowth. The level and impacts of Sox11 in brain with ICH remain indistinct. The major objective of the current work was to explore the spatiotemporal expression of Sox11 and its roles in secondary brain injury under the ICH impairment. The ICH rat model was established by injecting autologous blood into the right basal ganglia of male Sprague-Dawley rats. It was observed that Sox11 expression was notably elevated in brain tissue after ICH. The enhancement of Sox11 expression through miR-221 reduced neuronal apoptosis and inflammation in the affected rats. Furthermore, overexpression of Sox11 mitigated ICH-induced brain edema, blood-brain barrier disruption, and cognitive impairments. In contrast, Sox11 knockdown resulted in opposing effects. These findings highlight the crucial role of Sox11 in alleviating secondary brain injury following ICH. Thus, upregulating Sox11 presents a promising therapeutic strategy to reduce secondary brain injury in clinical ICH cases.

Keywords: Intracerebral hemorrhage; MiR-221; Neuroinflammation; Secondary brain injury; Sox11.

Fig. 1

The expression level of Sox11 increased after intracerebral hemorrhage. (A) Western blot analysis showed that Sox11 protein levels gradually rise and peak at 24 h after ICH. (B) Quantitative analysis of the protein levels of Sox11 which were normalized to the mean value of that in the Sham group. (C) Double-immunofluorescence analysis was performed with antibodies against Sox11 and NeuN (neuronal marker). The nuclei were fluorescently labeled with DAPI. Arrows point to Sox11-positive neurons. Scale bar = 20 μm. (D) Quantification of the relative fluorescence intensities. (E) qPCR shown that the relative expression level of miR-221 changed after ICH. **P < 0.01 compared with the Sham group. All values are expressed as mean ± SD, n = 6.

Fig. 2

mic-miR-221 and siRNA transfection effectively interfered with the expression of Sox11. (A) Western blot analysis showed that Sox11 protein levels are decreased in the Si-Sox11 treatment group but increased in the ICH + mic-miR-221 group. (B) Quantification of protein levels of Sox11 in rats in various groups. (C) Double-immunofluorescence analysis was performed with antibodies for Sox11 and NeuN (neuronal marker, red) and nuclei was fluorescently labeled with DAPI. Arrows point to Sox11-positive neurons. Scale bar = 20 μm. (D) Quantification of the relative fluorescence intensity of Sox11. ICH group compared with the Sham group; ICH + mic-miR-221 group compared with the ICH + Vector group; ICH + Si-Sox11 group compared with the ICH + Si-Control group. ** P < 0.01. All data are expressed as mean ± SD, n = 6.

Fig. 3

Sox11 effectively inhibits neuroinflammation. (A) Double-immunofluorescence analysis was performed with antibodies for microglia (green) and M2-microglia (red) and nuclei was fluorescently labeled with DAPI. Arrows point to Sox11-positive neurons. Scale bar = 20 μm. (B) Quantification of the percentage of M2-microglia. (C–H) The levels of TNF-α, IL-β and IL-13 in the serum and CSF were tested by ELISA. ICH group compared with the Sham group; ICH + mic-miR-221 group compared with the ICH + Vector group; ICH + Si-Sox11 group compared with the ICH + Si-Control group. ** P < 0.01 and * P < 0.05. All data are expressed as mean ± SD, n = 6.

Fig. 4

Sox alleviates brain damage by inhibiting nerve cell death. (A) Double immunofluorescence analysis for NeuN and TUNEL was performed. Nuclei were fluorescently labeled with DAPI. Arrows indicated apoptotic neurons (TUNEL- and NeuN-positive cells). Scale bar = 20 μm. (B) Fluoro-Jade C (FJC) staining was performed to assess neuronal degeneration in various groups at 72 h after ICH. Scale bar = 20 μm. (C) Percentages of TUNEL-positive neurons were shown. (D) Quantitative analysis of FJC staining in brain sections of each group. FJC-positive cells were counted per × 400 field. ICH group compared with the Sham group; ICH + mic-miR-221 group compared with the ICH + Vector group; ICH + Si-Sox11 group compared with the ICH + Si-Control group; **P < 0.01. All data are expressed as mean ± SD, n = 6.

Fig. 5

The protective effect of Sox11 on behavioral/cognitive dysfunction testing in the rat after ICH. (A) Representative images of swimming trajectories on day 34 was shown. (B) Motor learning was assessed by the escape latency (time, s). (C) Spatial learning was assessed by the length of the swimming track of each rat. (D) Motor ability is assessed by time spent on the rotating stick. (E) The assessment of sensory ability was conducted by measuring the time required to remove the sticker. (F) Balance is evaluated based on the ratio of errors made. (G) The neurological scores of rats in each group. ICH group compared with the Sham group; ICH + mic-miR-221 group compared with the ICH + Vector group; ICH + Si-Sox11 group compared with the ICH + Si-Control group; **P < 0.01 and *P < 0.05. All data are expressed as mean ± SD, n = 6.

Fig. 6

Increasing the expression level of Sox11 can maintain the integrity of the blood-brain barrier and inhibit brain edema. (A) The level of EB extravasation was increased in the rat brain after ICH, whereas it significantly reduced in mic-miR-221-treated rats. (B) The levels of ROS in the brain tissue of rats in various groups. (C) The levels of LDH in the CSF of rats in each group. (D) Brain water content was partially reduced by overexpression of Sox11. ICH group compared with the Sham group; ICH + mic-miR-221 group compared with the ICH + Vector group; ICH + Si-Sox11 group compared with the ICH + Si-Control group; **P < 0.01. All data are expressed as mean ± SD, n = 6.