HBO Alleviates Neural Stem Cell Pyroptosis via lncRNA-H19/miR-423-5p/NLRP3 Axis and Improves Neurogenesis after Oxygen Glucose Deprivation

Abstract

Due to the limited neurogenesis capacity, there has been a big challenge in better recovery from neurological dysfunction caused by stroke for a long time. Neural stem cell (NSC) programmed death is one of the unfavorable factors for neural regeneration after stroke. The types of death such as apoptosis and necroptosis have been deeply investigated while the pyroptosis of NSCs is not quite understood. Although it is well accepted that hyperbaric oxygen (HBO) alleviates the oxygen-glucose deprivation (OGD) injury after stroke and reduces programmed death of NSCs, whether NSC pyroptosis is involved in this process is still unknown. Therefore, this study is aimed at studying the potential effect of HBO treatment on NSC pyroptosis following OGD exposure, as well as its influence on NSC proliferation and differentiation in vitro. The results revealed that OGD increased NOD-like receptor protein 3 (NLRP3) expression to induce the pyroptotic death of NSCs, which was rescued by HBO treatment. And the upregulated lncRNA-H19 functioned as a molecular sponge of miR-423-5p to target NLRP3 for NSC pyroptosis following OGD. Most importantly, it was confirmed that HBO exerted protection of NSCs against pyroptosis by inhibiting lncRNA-H19/miR-423-5p/NLRP3 axis. Moreover, HBO restraint of lncRNA-H19-associated pyroptosis benefited the proliferation and neuronal differentiation of NSCs. It was concluded that HBO attenuated NSC pyroptosis via lncRNA-H19/miR-423-5p/NLRP3 axis and enhanced neurogenesis following OGD. The findings provide new insight into NSC programmed death and enlighten therapeutic strategy after stroke.

Figure 1

HBO inhibited the activation of NLRP3/Caspase-1/GSDMD signaling in NSCs following OGD. (a, b) NSC viability and release of LDH were tested following OGD exposure and HBO treatment in diverse pressure. (c) The protein expression of NLRP3, Caspase-1 p20, and GSDMD-N in Con, OGD, OGD+HBO, and OGD+HBO+Nig groups was determined by WB. (d–f) Quantitative analysis on the expression of NLRP3, Caspase-1 p20, and GSDMD-N protein in the four groups. (g, h) The level of IL-1β and IL-18 produced by NSCs in the four groups was measured by ELISA. ^∗P < 0.05 vs. the Con group. ^#P < 0.05 vs. the OGD group. ^†P < 0.05 vs. the OGD+HBO group.

Figure 2

HBO mitigated NLRP3-dependent pyroptosis of NSCs caused by OGD. (a) Representative immunofluorescence staining of NLRP3 and Nestin in NSCs of Con, OGD, OGD+HBO, and OGD+HBO+Nig groups. Nestin⁺: green; NLRP3⁺: red; DAPI⁺: blue. Scale bar = 10 μm. (b) Quantitative counting of NLRP3/Nestin colabeling cells in the four groups. ^∗P < 0.05 vs. the Con group. ^#P < 0.05 vs. the OGD group. ^†P < 0.05 vs. the OGD+HBO group.

Figure 3

Expression of lncRNA-H19, miR-423-5p, and NLRP3 in NSCs exposed to OGD was altered by HBO treatment. (a–d) Representative scanning electron microphotographs of pyroptotic NSCs in Con, OGD, OGD+HBO, and OGD+HBO+Nig groups. White arrow: GSDMD pores. Scale bar = 5 μm. (e–g) Statistical analysis on the expression of lncRNA-H19, miR-423-5p, and NLRP3 mRNA in the four groups detected by qPCR. ^∗P < 0.05 vs. the Con group. ^#P < 0.05 vs. the OGD group. ^†P < 0.05 vs. the OGD+HBO group. (h) Pearson's analysis on the correlation between lncRNA-H19 and miR-423-5p. (i) Pearson's analysis on the correlation between miR-423-5p and NLRP3 mRNA. (j) Pearson's analysis on the correlation between lncRNA-H19 and NLRP3 mRNA.

Figure 4

lncRNA-H19 functioned as a molecular sponge of miR-423-5p to target NLRP3. (a) The binding site between miR-423-5p and wild-type lncRNA-H19 (H19-WT) predicted by bioinformational tool LncBase V2; the mutant lncRNA-H19 (H19-MUT) sequence was shown as well. (b) Double luciferase assay was performed to verify the binding of lncRNA-H19 and miR-423-5p. ^∗P < 0.05 vs. the negative control group (miR-NC). (c) Expression of miR-423-5p in NSCs transfected with either siRNA-H19 or pcDNA-H19 or corresponding negative control (siRNA-NC or pcDNA-NC) was examined by qPCR. ^∗P < 0.05 vs. the Con group; ^#P < 0.05 vs. the siRNA-NC group; ^†P < 0.05 vs. the pcDNA-NC group. (d) The predicted binding site between miR-423-5p and the 3′UTR of wild-type NLRP3 (NLRP3-WT) obtained from bioinformational tool microT-CDS; the mutant sequence of NLRP3 (NLRP3-MUT) was also shown. (e) The binding of miR-423-5p and NLRP3 was examined by double luciferase assay. ^∗P < 0.05 vs. the miR-NC group. (f) Expression of NLRP3 mRNA in NSCs transfected with either miR-423-5p inhibitor or miR-423-5p mimic or corresponding negative control (miR-NC inhibitor or miR-NC mimic) was detected by qPCR. ^∗P < 0.05 vs. the Con group; ^#P < 0.05 vs. the miR-NC inhibitor group; ^†P < 0.05 vs. the miR-NC mimic group. (g, h) WB determination and statistical analysis of NLRP3 protein in NSCs transfected as above. ^∗P < 0.05 vs. the Con group; ^#P < 0.05 vs. the miR-NC inhibitor group; ^†P < 0.05 vs. the miR-NC mimic group.

Figure 5

lncRNA-H19/miR-423-5p/NLRP3 axis was involved in NSC pyroptosis following OGD. (a) NSCs transfected with either siRNA-NC or siRNA-H19 or siRNA-H19+miR-NC inhibitor or siRNA-H19+miR-423-5p inhibitor were exposed to OGD and followed by qPCR assay of NLRP3 mRNA. (b, c) WB detection and statistical analysis on the expression of NLRP3 protein in NSCs treated as above. (d, e) ELISA was performed to examine IL-1β and IL-18 released from NSCs which were treated with the same manners as above. ^∗P < 0.05 vs. the Con group; ^♦P < 0.05 vs. the OGD group; ^#P < 0.05 vs. the OGD+siRNA-NC group; ^∆P < 0.05 vs. the OGD+siRNA-H19 group; ^†P < 0.05 vs. the OGD+siRNA-H19+miR-423-5p inhibitor group.

Figure 6

HBO attenuated NSC pyroptosis by inhibiting lncRNA-H19/miR-423-5p/NLRP3 axis following OGD. (a) NSCs transfected with either pcDNA-NC or pcDNA-H19 or pcDNA-H19+miR-NC mimic or pcDNA-H19+miR-423-5p mimic were exposed to OGD and treated by HBO; the level of NLRP3 mRNA was analyzed with qPCR. (b) Expression of NLRP3, Caspase-1 p20, and GSDMD-N protein in NSCs treated as above was determined by WB. (c–e) Statistical analysis on the protein expression of NLRP3, Caspase-1 p20, and GSDMD-N in the above six groups. (f, g) The level of IL-1β and IL-18 released from NSCs in the above six groups was examined using ELISA. ^∗P < 0.05 vs. the OGD group; ^♦P < 0.05 vs. the OGD+HBO group; ^#P < 0.05 vs. the OGD+HBO+pcDNA-NC group; ^∆P < 0.05 vs. the OGD+HBO+pcDNA-H19 group; ^†P < 0.05 vs. the OGD+HBO+pcDNA-H19+miR-NC mimic group.

Figure 7

HBO restraint of lncRNA-H19-associated pyroptosis produced a favorable effect on NSC neurogenesis after OGD. (a) BrdU/DAPI colabeling staining was performed to investigate the proliferation of NSCs at 3 days following HBO treatment. TUJ1/DAPI and GFAP/DAPI colabeling staining were, respectively, performed to investigate the neuronal and astrocytic differentiation of NSCs at 14 days following HBO treatment. BrdU⁺: red; TUJ1⁺: red; GFAP⁺: green; DAPI⁺: blue. Scale bar = 20 μm. (b–d) Quantitative counting on the three kinds of colabeling cells in Con, OGD, OGD+HBO, OGD+HBO+pcDNA-H19, and OGD+HBO+pcDNA-H19+MCC950 group. ^∗P < 0.05 vs. the Con group; ^#P < 0.05 vs. the OGD group; ^∆P < 0.05 vs. the OGD+HBO group; ^†P < 0.05 vs. the OGD+HBO+pcDNA-H19 group.