Zinc finger E-Box binding homeobox 2 (ZEB2)-induced astrogliosis protected neuron from pyroptosis in cerebral ischemia and reperfusion injury

Abstract

Ischemia injury can cause cell death or impairment of neuron and astrocytes, and thus induce loss of nerve function. central nervous systems injury induces an aberrant activation of astrocytes called astrogliosis. Pyroptosis, which is a kind of programmed cell death, was proved play an important role in ischemia injury. Zinc Finger E-Box Binding Homeobox 2 (ZEB2) promoted neuron astrogliosis, which play a protected role in neuron regeneration. However, its precise mechanism remains unclear. This study investigated the mechanism of ZEB2 on astrogliosis and neuron regeneration after cerebral ischemia reperfusion condition. To confirm our hypothesis, Neurons and astrocytes were isolated from fetal Sprague Dawley rats, in vivo Middle Cerebral Artery Occlusion/reperfusion (MCAO/R) rat model and in vitro oxygen-glucose deprivation/reperfusion (OGD/R)-treated astrocytes and neurocytes model were constructed. Our results showed that ZEB2 was expressed in nucleus of astrocyte and upregulated after OGD/R induction, ZEB2 promoted astrogliosis. Further upregulation of ZEB2 promoted the astrogliosis, which promoted neuron proliferation and regeneration by decreased pyroptosis. Moreover, this finding was further confirmed in vivo MCAO/R rat model. Overexpression of ZEB2 promoted astrogliosis, which decreased infarct volume and accumulated recovery of neurological function by alleviated pyroptosis. This finding revealed that ZEB2 was a regulator of the astrogliosis after ischemia/reperfusion injury, and then astrogliosis promoted neuron regeneration via decreased neuron pyroptosis. Therefore, ZEB2 may be a potential therapeutic target for ischemia/reperfusion injury.

Keywords: Mcao/R; ZEB2; astrocyte; neural regeneration; pyroptosis.

Figure 1.

ZEB2 promoted the activation of astrocyte after OGD/R in vitro. A: Western blot assay of ZEB2 expression in astrocyte. B: Quantification of Western blot band intensity. C: Immunofluorescence staining for GFAP (green fluorescence), ZEB2 (red fluorescence), and DAPI (blue) in astrocytes. D: Fluorescence intensity of ZEB2. E: Fluorescence intensity of GFAP. E: Fluorescence intensity of ZEB2. E: Fluorescence intensity of GFAP. (Error bars represent mean ± SD, Magnification:400×; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Figure 2.

ZEB2 promoted astrogliosis after OGD/R in vitro. A: Western blot assay of ZEB2 and GFAP expression in astrocyte. B: Quantification of ZEB2 Western blot band intensity. C: Quantification of ZEB2 Western blot band intensity. (Error bars represent mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 3.

Astrogliosis promoted neuron regeneration in vitro. A: Immunofluorescence staining for NeuN+ (green fluorescence), BrdU+ (red fluorescence), and DAPI (blue fluorescence) in neurons. B: Immunofluorescence staining for BDNF (green fluorescence), MAP2+ (red fluorescence) and DAPI (blue fluorescence) in neurons. C: Fluorescence intensity of NeuN. D: Fluorescence intensity of BDNF. E: Fluorescence intensity of Brud. F: Fluorescence intensity of MAP2. G-H: Results of flow cytometry demonstrating astrogliosis alleviated apoptosis of neuron. I: Results of CCK8 demonstrating astrogliosis promoted proliferation of neuron (Error bars represent mean ± SD, Magnification:400×; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Figure 4.

Astrogliosis promoted neuron regeneration by decreasing pyroptosis in vitro. A: Western blot assay of pyroptosis relative protein expression in neuron. B: Quantification of Caspase-1 Western blot band intensity. C: Quantification of IL-1β Western blot band intensity. D: Quantification of NLRP3 Western blot band intensity. E: Quantification of GSDMD Western blot band intensity. F: Quantification of cleaved N-terminal GSDMD Western blot band intensity. G: ELISA analysis of the secretion of IL-1β in neuron. H: ELISA analysis of the secretion of IL-18 in neuron. (Error bars represent mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Figure 5.

Timeline of experimental procedures (MCAO, reperfusion, behavioral tests, and brain harvested) in MCAO/R rats

Figure 6.

ZEB2 expression after AAV injection and MCAO/R model establishment. A. Western blot assay was used to evaluate the infection of AAV. B. Quantification of ZEB2 Western blot band intensity in each groups

Figure 7.

Overexpression of ZEB2 in brain tissues of MCAO/R rats promoted neuron regeneration by alleviating pyroptosis. A: Representative track images of each group mice. B: Mean escape latency time during the orientation navigation test on 1, 3,7,14,21,28 days after MCAO/R. C: Time of the rats stay in the target quadrant on 28 days after MCAO/R. D: mNSS score on 1, 3,7,14,21,28 days after MCAO/R. E: Representative images of brain slices stained by TTC in different groups at 28 days after MCAO/R. F: Staining by Nissl showed the Nissl bodies of hippocampus and cerebral cortex. G: Staining by Luxol fast blue (LFB) showed the myelin of hippocampus and cerebral cortex. H: Western blot analysis of the expression of pyroptosis protein i.e., caspase-1, IL-1β, NLRP3, gasdermin, cleaved N-terminal GSDMD in brain tissue. I: Relative protein expression of caspase-1. J. Relative protein expression of IL-1β. K: Relative protein expression of NLRP3. L: Relative protein expression of gasdermin, M: Relative protein expression of cleaved N-terminal GSDMD. N: ELISA analysis of the expression of IL-1β in brain tissue of rats on 28 days after MCAO/R. O: ELISA analysis of the expression of IL-18 in brain tissue of rats on 28 days after MCAO/R. (Error bars represent mean ± SD, Magnification:400×; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001