IRE1α inhibition decreased TXNIP/NLRP3 inflammasome activation through miR-17-5p after neonatal hypoxic-ischemic brain injury in rats

Abstract

Background: The endoplasmic reticulum (ER) is responsible for the control of correct protein folding and protein function which is crucial for cell survival. However, under pathological conditions, such as hypoxia-ischemia (HI), there is an accumulation of unfolded proteins thereby triggering the unfolded protein response (UPR) and causing ER stress which is associated with activation of several stress sensor signaling pathways, one of them being the inositol requiring enzyme-1 alpha (IRE1α) signaling pathway. The UPR is regarded as a potential contributor to neuronal cell death and inflammation after HI. In the present study, we sought to investigate whether microRNA-17 (miR-17), a potential IRE1α ribonuclease (RNase) substrate, arbitrates downregulation of thioredoxin-interacting protein (TXNIP) and consequent NLRP3 inflammasome activation in the immature brain after HI injury and whether inhibition of IRE1α may attenuate inflammation via miR-17/TXNIP regulation.

Methods: Postnatal day 10 rat pups (n = 287) were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O₂). STF-083010, an IRE1α RNase inhibitor, was intranasally delivered at 1 h post-HI or followed by an additional one administration per day for 2 days. MiR-17-5p mimic or anti-miR-17-5p inhibitor was injected intracerebroventricularly at 48 h before HI. Infarct volume and body weight were used to evaluate the short-term effects while brain weight, gross and microscopic brain tissue morphologies, and neurobehavioral tests were conducted for the long-term evaluation. Western blots, immunofluorescence staining, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and co-immunoprecipitation (Co-IP) were used for mechanism studies.

Results: Endogenous phosphorylated IRE1α expression was significantly increased after HI. Intranasal administration of STF-083010 alleviated brain injury and improved neurological behavior. MiR-17-5p expression was reduced after HI, and this decrease was attenuated by STF-083010 treatment. MiR-17-5p mimic administration ameliorated TXNIP expression, NLRP3 inflammasome activation, caspase-1 cleavage, and IL-1β production, as well as brain infarct volume. Conversely, anti-miR-17-5p inhibitor reversed IRE1α inhibition-induced decrease in TXNIP expression and inflammasome activation, as well as exacerbated brain injury after HI.

Conclusions: IRE1a-induced UPR pathway may contribute to inflammatory activation and brain injury following neonatal HI. IRE1a activation, through decay of miR-17-5p, elevated TXNIP expression to activate NLRP3 inflammasome and aggravated brain damage.

Keywords: Hypoxia–ischemia; IRE1α; MicroRNA-17 (miR-17); Neonatal; Nod-like receptor protein 3 (NLRP3); Thioredoxin-interacting protein (TXNIP).

Fig. 1

Time course expression of pIRE1α in brain tissues and Immunofluorescence staining showing co-localization post HI. a Western blot assay for temporal profile of pIRE1α and IRE1α expressions from ipsilateral hemisphere at 0, 3, 6, 12, 24, and 72 h post HI; data are expressed as mean ± SEM, n = 5 per group, per time point. Relative densities have been normalized against the sham group. *P < 0.05 compared with sham group. b–d Representative microphotographs of immunofluorescence staining for pIRE1α (red) co-localization on neurons (NeuN, green; b), microglia (Iba-1, green; c), and astrocytes (GFAP, green; d) at 6 h post HI. n = 3 per group, scale bar = 100 μm

Fig. 2

STF-083010, an IRE1α inhibitor, reduced brain infarction at 24 and 72 h post HI. a–c Representative photographs of TTC-stained coronal brain sections (a), quantitative analysis of infarct volume (b), and body weight gain (c) with single-dose treatment at 24 h post HI. d–f. Representative photographs of TTC-stained coronal brain sections (d), quantitative analysis of infarct volume (e), and body weight gain (f) with multi-dose treatment at 72 h post HI. Data are represented as mean ± SEM, n = 6 in each group. **P < 0.01 compared with sham group, ^##P < 0.01 compared with HI group, ^#P < 0.05 compared with HI group, ^§§P < 0.01 compared with vehicle+HI group, and ^&P < 0.05 compared with STF-15+HI group. STF: STF-083010, an IRE1α RNase-specific inhibitor. STF-15: STF-083010 (15 μg/pup). STF-45: STF-083010 (45 μg/pup)

Fig. 3

STF-083010 ameliorated brain atrophy and improved physical development at 6 weeks post HI. a–c Quantification of right (ipsilateral) to left (contralateral) hemispheric (RH/LH) weight ratio showed that brain atrophy was attenuated after treatment (a), statistical analyses of residual brain volume showed that treatment significantly attenuated HI induced brain tissue loss (b) and top view of the brains and brain slices with Nissl staining at 6 weeks post HI (c). d–f Body weight gain (d), quantification of organ (heart, lung, liver, spleen, and kidney) to body weight ratios showed no significant difference between groups (e) and representative photographs of organs (f). Data are expressed as mean ± SEM, n = 8 in sham or vehicle+HI groups and 9 in STF + HI group. **P < 0.01 compared with sham group, *P < 0.05 compared with sham group, ^##P < 0.01 compared with vehicle+HI group, and ^#P < 0.05 compared with vehicle+HI group

Fig. 4

STF-083010 improved long-term neurobehavioral function at 5 and 6 weeks post HI. All animals in the HI group exhibited severe neurobehavioral impairments in the sensorimotor tests (a), foot fault test (b), rotarod test (c), T-maze test (d), and Morris water maze test (e–g). STF-083010 treatment significantly improved neurological outcomes in the above tests except for foot fault test (b). Data are expressed as mean ± SEM, n = 8 in sham or vehicle+HI groups and 9 in STF+HI group. **P < 0.01 compared with sham group, *P < 0.01 compared with sham group, ^##P < 0.01 compared with vehicle+HI group, ^#P < 0.05 compared with vehicle+HI group

Fig. 5

IRE1α inhibition upregulated miR-17-5p expression post HI and. TXNIP is a target of miR-17-5p. a qPCR results showed the down-regulation of miR-17-5p in HI compared with naive group. (n = 4, *P < 0.05 compared with naive group). b q-PCR results showed IRE1α inhibition upregulated mir-17-5p expression at 6 h after HI. (n = 4, **P < 0.01 compared with sham group, *P < 0.05 compared with sham group, and ^#P < 0.05 compared with vehicle+HI group). c Sequence alignment showed putative miR-17-5p binding sites within the 3′-UTR of the TXNIP mRNA in rats. d, f The expression levels of TXNIP mRNA (d, n = 3) and protein (f, n = 4, *P < 0.05 compared with negative control or naive group) were reduced at 48 h after administration of miR-17-5p mimic. miR-17-5p mimic-0.05 or 0.5: Syn-rno-miR-17-5p miScript miRNA mimic (0.05 or 0.5 nmol/pup). e, g The expression levels of TXNIP mRNA (e, n = 3) and protein (g, n = 4, *P < 0.05 compared with negative control or naive group) were increased at 48 h after administration of miR-17-5p inhibitor. miR-17-5p inhibitor-0.1 or 1: Anti-rno-miR-17-5p miScript miRNA inhibitor (0.1 or 1 nmol/pup)

Fig. 6

miR-17-5p mimic attenuated infarction, downregulated TXNIP expression, NLRP3 inflammasome activation and IL-1β production at 24 h post HI. a, b Representative photographs of TTC-stained coronal brain sections (a) and quantitative analysis of infarct volume (b) at 24 h post HI. c–f Representative western blotting bands (c) and quantification (d–f) of TXNIP, cleaved caspase-1, and IL-1β expressions. g Representative Co-IP assay bands for interaction between NLRP3 and TXNIP. Data are expressed as mean ± SEM, n = 6 in each group. **P < 0.01 compared with sham group, *P < 0.05 compared with sham group, ^##P < 0.01 compared with HI group, ^#P < 0.05 compared with HI group, ^§§P < 0.01 compared with NC + HI group, and ^§P < 0.05 compared with NC+HI group. NC negative control

Fig. 7

miR-17-5p inhibitor increased infarction, upregulated TXNIP expression, NLRP3 inflammasome activation and IL-1β production at 24 h post HI. a, b Representative photographs of TTC-stained coronal brain sections (a) and quantitative analysis of infarct volume (b) at 24 h post HI. c–f Representative western blotting bands (c) and quantification (d–f) of TXNIP, cleaved caspase-1 and IL-1β expressions. g Representative Co-IP assay bands for interaction between NLRP3 and TXNIP. Data are expressed as mean ± SEM, n = 6 in each group. **P < 0.01 compared with sham group, *P < 0.05 compared with sham group, ^##P < 0.01 compared with vehicle+HI group, ^#P < 0.05 compared with vehicle+HI group, ^§§P < 0.01 compared with STF+HI group, ^§P < 0.05 compared with STF+HI group, ^&&P < 0.01 compared with STF+NC+HI group, and ^&P < 0.05 compared with STF+NC+HI group