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. 2019 Oct 27;8(11):1326.
doi: 10.3390/cells8111326.

Over-Activated Proteasome Mediates Neuroinflammation on Acute Intracerebral Hemorrhage in Rats

Hock-Kean Liew  1   2   3   4 , Wei-Fen Hu  1   4 , Peter Bor-Chian Lin  5 , Po-Kai Wang  6   7 , Andy Po-Yi Tsai  5 , Cheng-Yoong Pang  1   2   3 , Tsung-Ying Chen  1   6   7   8
Affiliations

Over-Activated Proteasome Mediates Neuroinflammation on Acute Intracerebral Hemorrhage in Rats

Hock-Kean Liew et al. Cells. .

Abstract

Background: Neuroinflammation is a hallmark in intracerebral hemorrhage (ICH) that induces secondary brain injury, leading to neuronal cell death. ER stress-triggered apoptosis and proteostasis disruption caused neuroinflammation to play an important role in various neurological disorders. The consequences of ER stress and proteostasis disruption have rarely been studied during the course of ICH development.

Methods: ICH was induced by collagenase VII-S intrastriatal infusion. Animals were sacrificed at 0, 3, 6, 24, and 72 h post-ICH. Rats were determined for body weight changes, hematoma volume, and neurological deficits. Brain tissues were harvested for molecular signaling analysis either for ELISA, immunoblotting, immunoprecipitation, RT-qPCR, protein aggregation, or for histological examination. A non-selective proteasome inhibitor, MG132, was administered into the right striatum three hours prior to ICH induction.

Results: ICH-induced acute proteasome over-activation caused the early degradation of the endoplasmic reticulum (ER) chaperone GRP78 and IκB protein. These exacerbations were accompanied by the elevation of pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP) and pro-inflammatory cytokines expression via nuclear factor-kappa B (NF-κB) signal activation. Pre-treatment with proteasome inhibitor MG132 significantly ameliorated the ICH-induced ER stress/proteostasis disruption, pro-inflammatory cytokines, neuronal cells apoptosis, and neurological deficits.

Conclusions: ICH induced rapid proteasome over-activation, leading to an exaggeration of the ER stress/proteostasis disruption, and neuroinflammation might be a critical event in acute ICH pathology.

Keywords: ER stress; GRP78; NFκB; intracerebral hemorrhage; oxidative stress; proteasome activity; protein aggregation; proteostasis disturbance; ubiquitination.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MG132 pre-treatment reduced hematoma volume, oxidative stress, body weight loss, and neurological impairments of intracerebral hemorrhage (ICH) rats. Representative image of hemorrhagic brain sections in ICH and MG132 pre-treated-ICH rats at 3, 24, and 72 h post-ICH injury (A). Statistical hematoma expansion volume (B), protein carbonyl content (C), body weight change (D), and modified Neurological Severity Score (mNSS) neurobehavioral assay (E) at 0 (normal), 3, 24, and 72 h post-ICH; respectively. Values are indicated by means ± SEM; n = 6 each group, * p < 0.05; *** p < 0.001; compared to the normal group; # p < 0.05; ### p < 0.001; compared to the Saline + ICH group, respectively.
Figure 2
Figure 2
Expression of ubiquitinated protein in ICH injury striatum. Ubiquitin protein levels evaluated by ubiquitin competitive enzyme-linked immunosorbent assay at normal, 3, 24, and 72 h post-ICH injury (A). Rat brains were stained for ubiquitin (green) and dual stained with OX-42 (anti-CD11b/c; microglia, red), RECA (anti-rat endothelial cells antigen; vascular endothelial cells, red), NeuN (anti-neuronal nuclei; neuron, red) and GFAP (anti-glial fibrillary acidic protein; astrocyte, red) in the ipsilateral striatal (3 h post-ICH) (B). Value are indicated by means ± SEM; n = 6 each group, * p < 0.05; *** p < 0.001; compared to normal group; # p < 0.05; ### p < 0.001; compared to Saline + ICH group, respectively.
Figure 3
Figure 3
ICH induced acute proteasome over-activation. The proteasome activity was measured by fluorometric assay in ipsilateral striatal lysates at normal, 3, 24, and 72 h post-ICH. Values are indicated by means ± SEM; n = 6 each group, * p < 0.05; *** p < 0.001; compared to the normal group; ### p < 0.001; compared to the Saline + ICH group, respectively.
Figure 4
Figure 4
Ubiquitinated protein aggregates in ICH striatal. Representative naïve SDS-PAGE of ubiquitinylated protein accumulation (A) and statistical analysis of high molecular weight (>50 kDa) polyubiqutinated protein aggregation in normal, 3, 24, 72 h post-ICH injury, n = 6 each group (B). Transmission electron microscopic pictures of neuronal stroma stained with uranyl acetate and lead citrate in region of contralateral striatum (I), perihematomal striatal (II and III) and core (IV) at 3 h post-ICH. ER, endoplasmic reticulum; Ly, lysosome; M, mitochondria; N, nucleus; abnormal protein aggregated (arrow point) (C). Values are indicated by means ± SEM; * p < 0.05; compared to the normal group; # p < 0.05; compared to the Saline + ICH group, respectively (B).
Figure 5
Figure 5
Effects of MG132 pretreatment on GRP78, CHOP, IκB, and pNF-κB protein levels after ICH injury. Representative Western blotting images showing the 78-kDa band of GRP78, 19-kDa band of CHOP (A), and pNF-κB, and IκB (D). Quantitative analyses of GRP78 (B) and CHOP (C) protein detected in the ipsilateral and contralateral striatum lysate from normal, Saline + ICH-3h, MG132 + ICH-3h, Saline + ICH-24 h, MG132 + ICH-24h, Saline + ICH-72h, and MG132 + ICH-72h animals, respectively. Quantitative analyses of IκB (E) and pNF-κB (F) protein detected in the ipsilateral striatum lysate from normal, Saline + ICH-3h, and MG132 + ICH-3h animals, respectively. Values are indicated by means ± SEM; n = 6 each group, * p < 0.05; *** p < 0.001; compared to the normal group; # p < 0.05; compared to the Saline + ICH group, respectively.
Figure 6
Figure 6
ICH induced the protein degradation of GRP78 and IκB protein. Representative Western blotting analysis of striatal tissue lysates immunoprecipitated with anti-glucose-regulated protein 78 kDa (GRP78) (A) and anti-IκB (C) antibodies followed by anti-ubiquitin antibody at normal (NC), 3 (ICH-3h), and 72 h (ICH-72h) post-ICH injury. Value of quantitative of relative ubiquitinated GRP78 (B) and IκB (D) are indicated by means ± SEM; n = 4 each group, * p < 0.05; *** p < 0.001; compared to the normal group, respectively.
Figure 7
Figure 7
The mRNA expression of GRP78, CHOP, sXBP1/XBP1 ratio, and ATF4 in ipsilateral striatum. The RT-qPCR measured the mRNA levels for GRP78 (A), CHOP (B), sXBP1/XBP1 (C), and ATF4 (D) in the ipsilateral striatum at normal control (NC), 3 h, 6 h, 24 h, and 72 h post-ICH injury. Values are indicated by means ± SEM; n = 6 each group, * p < 0.05; ** p < 0.01; *** p < 0.001; compared to the normal control group, respectively.
Figure 8
Figure 8
Immunohistochemical staining of CHOP expression at 1 day post-ICH. Yellowish color in the merges (a4, b4, and c4) indicates the co-localization of CHOP expression in neurons (NeuN, a1–a5), microglias (OX-42, b1–b5), and endothelial cells (RECA, c1–c5) in Saline + ICH (A) and MG132 + ICH (B), respectively.
Figure 9
Figure 9
Expression levels of cytokines measured in the ipsilateral striatal tissues. The tumor necrosis factor-α (TNF-α) (A), interleukin (IL)-6 (B), IL-1β (C), and IL-10 (D) in normal, 3 h, 24 h, and 72 h post-ICH. Value are indicated by means ± SEM; n = 8 each group, * p < 0.05; *** p < 0.001; compared to normal group; # p < 0.05; ### p < 0.001; compared to Saline + ICH group, respectively.
Figure 10
Figure 10
Neuronal apoptosis in hemorrhagic striatal rat at 1 day post-ICH injury. Representative TUNEL-positive staining of apoptotic cells in four regions around the perihematomal area (A, I–IV). Statistical measurement of apoptotic cells subjected to ICH injury in the Saline + ICH and MG132 + ICH groups (B), respectively. Representative TUNEL-positive image of Saline + ICH (C) and MG132 + ICH (D), respectively. Values are indicated by means ± SEM; n = 4 each group, # p < 0.05; as compared to the Saline + ICH group. TUNEL staining was conducted following GRP78 (a), CHOP (b), RECA (c), OX-42 (d) and NeuN (e) on the brain sections of the perihematomal area of ICH striatal (E).
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References

    1. Keep R.F., Hua Y., Xi G. Intracerebral haemorrhage: Mechanisms of injury and therapeutic targets. Lancet Neurol. 2012;11:720–731. doi: 10.1016/S1474-4422(12)70104-7. - DOI - PMC - PubMed
    1. Duan X., Wen Z., Shen H., Shen M., Chen G. Intracerebral Hemorrhage, Oxidative Stress, and Antioxidant Therapy. Oxid. Med. Cell. Longev. 2016;2016:1203285. doi: 10.1155/2016/1203285. - DOI - PMC - PubMed
    1. Hoff J.T., Xi G. Brain edema from intracerebral hemorrhage. Acta Neurochir. Suppl. 2003;86:11–15. - PubMed
    1. Hu X., Tao C., Gan Q., Zheng J., Li H., You C. Oxidative Stress in Intracerebral Hemorrhage: Sources, Mechanisms, and Therapeutic Targets. Oxid. Med. Cell. Longev. 2016;2016:3215391. doi: 10.1155/2016/3215391. - DOI - PMC - PubMed
    1. Kazui S., Naritomi H., Yamamoto H., Sawada T., Yamaguchi T. Enlargement of spontaneous intracerebral hemorrhage. Incidence and time course. Stroke. 1996;27:1783–1787. doi: 10.1161/01.STR.27.10.1783. - DOI - PubMed

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