Hemoglobin Derived from Subarachnoid Hemorrhage-Induced Pyroptosis of Neural Stem Cells via ROS/NLRP3/GSDMD Pathway

Abstract

Accumulating evidence has demonstrated that neural stem cells (NSCs) have regenerative capacity after brain injuries, such as in aneurysmal subarachnoid hemorrhage (SAH). The reactive oxygen species (ROS)-induced NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome triggers inflammatory responses and pyroptosis of cells; however, whether ROS-induced neuroinflammation modulates the fate of endogenous NSCs after SAH remains largely unknown. In this study, the level of IL-1β was increased in the cerebrospinal fluid (CSF) of patients with SAH. In an endovascular perforation model of SAH in mice, the secretion of IL-1β increased to a peak at 24 h following SAH, and the expression of Caspase1 and NLRP3 was elevated in the hippocampus. Primary cultured NSCs were incubated with hemoglobin (Hb) to mimic SAH in vitro. The cell viability, LDH release, intracellular ROS levels, scanning electron microscopy (SEM), and the expression of NLRP3 and pyroptosis indicators (GSDMD, ASC, and Caspase-1) in NSCs after SAH were examined to investigate the process of pyroptosis. We found that pyroptotic death featuring cellular swelling, cell membrane pore formation and elevated IL-1β was increased in cultured primary NSCs after Hb treatment, as was the expression of NLRP3, ASC, Caspase-1, and GSDMD. In addition, we found that ROS-induced pyroptosis of NSCs by activating the NLRP3/GSDMD pathway. These findings suggest that pyroptosis of NSCs induced by Hb can impede neural regeneration after SAH.

Figure 1

Elevated IL-1β levels of CSF in SAH patients. (a) The IL-1β of CSF samples in control and SAH patients were determined by ELISA. The mean IL-1β level of all CSF samples including day 1-3 after SAH was significantly higher than that of control samples. (b) The IL-1β level of CSF in SAH patients at day 1 (n = 7) was significantly higher than that at day 2 (n = 14) and day 3 (n = 12) after SAH. Data were presented as the Mean ± SD. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 vs. control group; ###p < 0.001 vs. SAH at day 1 group.

Figure 2

NLRP3 mediated pyroptosis in rat hippocampus after SAH. (a) Representative images of the rat brains in control and SAH group after operation. (b) The SAH grade scores of the rats in control and SAH groups. (c) IL-1β expression in hippocampus were detected by ELISA. (d) Representative IF images of NLRP3 and Caspase1 p20 in hippocampus of rats after SAH. Scale bar = 50 μm. Data were expressed as Mean ± SD (n = 6), ^∗∗∗p < 0.001 vs. control group.

Figure 3

Hb triggered pyroptosis in primary NSCs in vitro. (a–c) Cell survival curve (a), cell viability (b), and LDH release (c) of primary NSCs treated by 25 μM Hb at different time points. (d) IL-1β protein secreted in the supernatant of NSCs after Hb treatment using ELISA. (e) Representative images of Nestin immunofluorescence staining in NSCs, which displayed pyroptotic typical swelling at 24 h after Hb treatment. Scale bar = 50 μm. (f) Representative scanning electron microscopy (SEM) images of NSCs at 24 h showed more pore formations on membrane on NSCs as white arrows in Hb treatment group. Scale bar = 2 μm. Data were expressed as Mean ± SD (n = 4), ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs. control group.

Figure 4

The NLRP3/GSDMD mediated the pyroptosis in primary NSCs exposed to Hb. (a) Hb treatment induced the elevated mRNA expressions of NLRP3, ASC, and Caspase1 in NSCs using qRT-PCR assay. (b) Western blot analysis showed the upregulated protein expressions of NLRP3, ASC, Cleaved Caspase1, and Cleaved GSDMD in Hb-treated NSCs. The band densities in western blot analysis were quantified using Image J Software. (c) Representative IFs staining images of NLRP3,Caspase1 P20, and GSDMD in NSCs with or without Hb treatment at 24 h. Scale bars = 50 μm. Data were expressed as Mean ± SD (n = 4), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs. control group.

Figure 5

Effects of ROS on Hb-induced pyroptosis in primary NSCs. (a) Hb treatment significantly induced ROS production in intracellular of primary NSCs by FCM analsysis. (b) The Hb-induced inhibition of cell viability in NSCs was retrieved by NAC pretreatment (a ROS inhibitor). (c) NAC prestreatment restored Hb-induced LDH release in NSCs. (d) Secreted IL-1β in the supernatant was decreased in NSCs when pretreated with NAC. (e) NAC pretreatment blocked the Hb-induced ROS production in intracellular of primary NSCs. (f) Representative images Nestin immunofluorescence stainingin NSCs and the cell body area was analyzed using Image J Software. Scale bar = 50 μm. Data were expressed as Mean ± SD (n = 3). ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared with the control group. #p < 0.05, ###p < 0.001 vs. Hb treatment group.

Figure 6

ROS scavenger mitigated activation of NLRP3/GSDMD pathway in Hb-induced pyroptosis of NSCs. (a) Hb-induced upregulated mRNA levels of NLRP3, ASC, and Caspase 1 were restored by NAC pretreatment by qRT-PCR assay (b) Western bolt analysis showed that NAC pretreatment blocked the upregulated protein levels of NLRP3, Caspase 1, Cleaved Caspase 1, Cleaved GSDMD, and ASC by Hb in NSCs. (c) Representative images of NLRP3 and GSDMD immunostainings in NSCs. Scale bars = 50 μm. Data were expressed as Mean ± SD (n = 3).^∗∗p < 0.01, ^∗∗∗p < 0.001 compared with the control group. #p < 0.05, ###p < 0.001 vs. Hb treatment group.

Figure 7

Proposed model of pyroptosis induced by hemoglobin via ROS/NLRP3 axis in NSCs.