DHEA Attenuates Microglial Activation via Induction of JMJD3 in Experimental Subarachnoid Haemorrhage

Figures

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  Fig. 1. DHEA mitigated severe neuronal damage after endovascular perforation in SAH model mice. a After intraperitoneal injection, DHEA levels in the serum, cortex, hippocampus and hypothalamus were measured by ELISA (n = 5, one-way ANOVA, *P < 0.05 compared with serum in the vehicle group; #P < 0.05, ##P < 0.01 compared with serum in the SAH + DHEA group). b, c DHEA treatment resulted in less water content on POD 1 and better neurological test performance on both POD 1 and POD 7 (n = 6 in both experiments, one-way ANOVA). d, e Representative TUNEL images 24 h after SAH and whole-brain NeuN+ TUNEL+/ NeuN+ proportion (n = 3 brains, one-way ANOVA). POD, postoperative day; Green, NeuN; red, TUNEL; bar = 1 mm. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 2. DHEA attenuated excessive inflammation after SAH in mice. The mRNA levels of IL-1β, IL-6, TNF-α, IL-12, CCL2 and NLRP3 from affected brain extracts were detected by qPCR (n = 5, one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001)

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  Fig. 3. DHEA exerted an anti-inflammatory effect by regulating microglial activation in vivo and in vitro. Co-localization of Iba-1 and CD86 or CD206 demonstrated a switch from proinflammatory to anti-inflammatory activation in the presence of a DHEA after SAH in the piriform and b Hb exposure in vitro. Magnified views are marked with dashed line boxes. c TNF-α levels in the CM from various treatment microglia was tested by ELISA (n = 4, t test between Hb and Hb + DHEA). d The positive cell count (histogram, left Y-axis) and positive cell proportion (polygram, right Y-axis) of Iba-1+ and CD86+/CD206+ from b piriform and c in vitro (n = 6 fields, 400 × 400 μm, t test for comparison between SAH/Hb and SAH/Hb + DHEA; *P, #P, §P < 0.05, **P, ##P, §§P < 0.01, ***P, ###P, §§§P < 0.001, ns, not significant). Hb, haemoglobin; Green, Iba-1; red, CD86; cyan, CD206; blue, DAPI; bar = 70 μm

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  Fig. 4. Transcription and translation of JMJD3 were induced by DHEA after Hb exposure. a, b Co-localization of Iba-1 and JMJD3 demonstrated an induction of JMJD3 expression after SAH/Hb exposure that was even higher in the presence of DHEA. Magnified views are marked with dashed line boxes. Green, Iba-1; red, JMJD3; blue, DAPI; bar = 70 μm. c The relative single cell fluorescence intensity (JMJD3 in Iba-1+/mean of JMJD3 in Iba-1-) was calculated and presented in a violin plot (n = 20 in sham, n = 79 in SAH, n = 43 in SAH + DHEA from 6 fields, non-specific one-way ANOVA, ****P < 0.0001). d Fluorescence intensity of JMJD3 after Hb exposure (n = 6 fields, one-way ANOVA). e, f qPCR and WB showed an induction of Jmjd3 mRNA and protein expression in microglia treated with DHEA (n = 4 in qPCR and n = 3 in WB, one-way ANOVA), ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Eth., ethanol

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  Fig. 5. DHEA alleviated H3K27me3, and this effect was blocked with a JMJD3 inhibitor in vitro. a, b Methylation level at H3K27 is indicated by WB (n = 4, one-way ANOVA, ns, not significant, *P < 0.05)

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  Fig. 6. JMJD3 mediated the anti-inflammatory effect of DHEA. The mRNA expression levels of a IL-1β, IL-6, TNF-α, and CCL2 and b two homeostasis genes were detected by qPCR (n = 5, non-parametric test for P2RY12 and one-way ANOVA for the other). The gene expression levels in the vehicle group treated with dimethyl sulfoxide (DMSO) or ethanol were set as 1. c The CM collected from microglia was added to the primary neurons for another 24 h, and neuron viability was assessed by CCK8 (n = 5, one-way ANOVA). d Representative fields of primary neurons cultured with CM from microglia exposed to different treatments for 24 h. e Neuronal morphology was investigated with Sholl analysis (n = 5, two-way ANOVA with Dunnett’s multiple comparisons test, the footnote* indicates the statistically significant difference between Hb and Hb + DHEA; the difference between vehicle and Hb is not shown; no significant difference between Hb + DHEA and Hb + DHEA + GSK-J4). Bar = 50 μm. Eth., ethanol. *P < 0.05, **P < 0.01

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  Fig. 7. DHEA promoted microglial anti-inflammatory activation after Hb exposure. mRNA expression of a Arg1, CD206 and b Irf4 and Irf7 were analysed by qPCR (n = 5, one-way ANOVA). c, d The protein levels were analysed by WB (n = 4, one-way ANOVA). Eth., ethanol; DMSO, dimethyl sulfoxide. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 8. The TrkA/Akt pathway was involved in the anti-inflammatory effects of DHEA in microglia. a, c JMJD3, TrkA/phosphorylated TrKA (pTrkA), Akt/pAkt and iNOS protein expression were analysed by WB (n = 3, one-way ANOVA). b mRNA levels of TNF-α, IL-10 and Tgf-β1 were analysed by qPCR (n = 3, one-way ANOVA). Eth., ethanol, DMSO, dimethyl sulfoxide. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 9. As innate immune cells in the CNS, microglia are activated after SAH in vivo and in vitro. Microglia then initiate an inflammatory cascade to aggravate neuronal damage, which is an important cause of EBI. A neurosteroid, DHEA, exerts an anti-inflammatory effect after Hb exposure by initiating the TrkA/Akt signalling pathway and inducing the expression of the H3K27me3 demethylase, Jmjd3. Jmjd3 then negatively regulates proinflammatory genes and modulates the proinflammatory/anti-inflammatory switch, possibly through its downstream transcription factors