International Journal of
Molecular Sciences Article
Thioredoxin-Interacting Protein Mediates Apoptosis in Early Brain Injury after Subarachnoid Haemorrhage Qing Zhao, Xudong Che, Hongxia Zhang, Guanping Tan, Liu Liu, Dengzhi Jiang, Jun Zhao, Xiang Xiang, Xiaochuan Sun and Zhaohui He * Department of Neurosurgery, the First Affiliated Hospital of Chongqing Medical University, 1 Friendship Road, Chongqing 400016, China;
[email protected] (Q.Z.);
[email protected] (X.C.);
[email protected] (H.Z.);
[email protected] (G.T.);
[email protected] (L.L.);
[email protected] (D.J.);
[email protected] (J.Z.);
[email protected] (X.X.);
[email protected] (X.S.) * Correspondence:
[email protected]; Fax: +86-23-6408-5875 Academic Editor: Christo Christov Received: 20 February 2017; Accepted: 13 April 2017; Published: 18 April 2017
Abstract: Early brain injury (EBI) is considered to be the major factor associated with high morbidity and mortality after subarachnoid haemorrhage (SAH). Apoptosis is the major pathological mechanism of EBI, and its pathogenesis has not been fully clarified. Here, we report that thioredoxin-interacting protein (TXNIP), which is induced by protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK), participates in EBI by promoting apoptosis. By using adult male Sprague-Dawley rats to establish SAH models, as well as Terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining, immunofluorescence, and western blot, we found that TXNIP expression significantly increased after SAH in comparison to the sham group and peaked at 48 h (up to 3.2-fold). Meanwhile, TXNIP was widely expressed in neurons and colocalized with TUNEL-positive cells in the hippocampus and cortex of SAH rats. After administration of TXNIP inhibitor-resveratrol (60 mg/kg), TXNIP small interfering RNA (siRNA) and the PERK inhibitor GSK2656157, TXNIP expression was significantly reduced, accompanied by an attenuation of apoptosis and prognostic indicators, including SAH grade, neurological deficits, brain water content, and blood-brain barrier (BBB) permeability. Collectively, these results suggest that TXNIP may participate in EBI after SAH by mediating apoptosis. The blockage of TXNIP induced by PERK could be a potential therapeutic strategy for SAH treatment. Keywords: thioredoxin-interacting protein; subarachnoid haemorrhage; early brain injury; apoptosis; protein kinase RNA-like ER kinase
1. Introduction Early brain injury (EBI) refers to the direct brain damage that occurs within 72 h after bleeding and is considered to be the major factor of poor prognosis after subarachnoid haemorrhage (SAH) [1]. Previous studies have confirmed that brain cell apoptosis occurs after SAH and that the levels of numerous apoptotic proteins increased during this process [2,3]. Apoptosis has gradually become regarded as the most important pathological event in aggravating brain cell death, blood-brain barrier (BBB) disruption, and brain edema after SAH [1]. Although some anti-apoptotic strategies have been developed in EBI treatment, the mortality and disability of SAH still remain high, indicating that there may be some unknown mechanisms.
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Thioredoxin-interacting protein (TXNIP), also named thioredoxin binding protein-2 or vitamin D3 upregulated is widely expressed in the nucleus, mitochondria, and cytoplasm of normal Int. J. Mol. Sci. 2017, protein-1, 18, 854 2 of 16 tissue cells [4]. TXNIP directly binds to the active cysteine residue of thioredoxin (TRX) under under pathological conditions and disturbs further disturbs the TRX/apoptosis signal-regulating pathological conditions and further the TRX/apoptosis signal-regulating kinase-1kinase-1 (ASK-1) (ASK-1) complex. Thisleads disruption release activation of the inhibitoryinhibitory complex. This disruption to ASK-1 leads releaseto andASK-1 activation of theand downstream cascade of downstream the apoptosis signallingprevious pathway [5]. Meanwhile, previous have the apoptosis cascade signallingofpathway [5]. Meanwhile, studies have found that TXNIPstudies participates found that TXNIP participates in ischaemic diseases [6,7]. is or important during EBI, but in ischaemic diseases [6,7]. Apoptosis is important during EBI,Apoptosis but whether not TXNIP participates in whether or not TXNIP participates in EBI by mediating apoptosis has not been explored. We EBI by mediating apoptosis has not been explored. We previously found that silencing the downstream previously found that silencing the downstream proteinreticulum of the protein protein of the protein kinase RNA-like endoplasmic (ER)kinase kinaseRNA-like (PERK) endoplasmic downstream reticulum (ER)reduce kinase neuronal (PERK) downstream protein reduce neuronalresearchers apoptosis after [3]. protein could apoptosis after SAHcould [3]. Coincidentally, haveSAH recently Coincidentally, researchers have recently discovered that PERK, a transmembrane protein in the ER, discovered that PERK, a transmembrane protein in the ER, significantly promotes TXNIP expression [8]. significantly TXNIP expression [8]. According these observations, weexpression hypothesized According topromotes these observations, we hypothesized thattoPERK-induced TXNIP maythat be PERK-induced may be involved in EBI through its apro-apoptotic function. involved in EBI TXNIP throughexpression its pro-apoptotic function. Therefore, we utilized TXNIP inhibitor, small Therefore, a TXNIP inhibitor, small interfering RNAthe (siRNA), PERK-specific interfering we RNAutilized (siRNA), and PERK-specific inhibitor to suppress activity and of TXNIP or PERK inhibitor to that suppress the activity TXNIP or PERK to confirm that the inhibition of PERK-induced to confirm the inhibition ofofPERK-induced TXNIP expression may exert anti-apoptotic and TXNIP expression may exert anti-apoptotic and neuroprotective effects after SAH. neuroprotective effects after SAH. 2. Results 2.1. TXNIP TXNIP Expression Expression Increased Increased Obviously Obviously after after SAH 2.1. SAH To confirm Here, rats rats were were To confirm our our speculation, speculation, we we designed designed the the experiments experiments shown shown in in Figure Figure 1. 1. Here, divided into different groups, as described in the methods below: sham, SAH, SAH + control divided into different groups, as described in the methods below: sham, SAH, SAH + control siRNA siRNA (Control), TXNIP siRNA, SAH + saline normal(NS), salineSAH (NS), SAH + resveratrol (RES), (Control), SAH + SAH TXNIP+ siRNA, SAH + normal + resveratrol (RES), SAH + SAH + dimethyl sulfoxide (DMSO, as a vehicle), and SAH + GSK2656157 (GSK). Both siRNA and dimethyl sulfoxide (DMSO, as a vehicle), and SAH + GSK2656157 (GSK). Both siRNA and GSK2656157 were were injected injected through through intracerebroventricular intracerebroventricular infusion, infusion, and and RES RES was was administered administered via via GSK2656157 intraperitoneal injection. intraperitoneal injection.
Figure 1. Experimental design (A–C). SD rats, Sprague–Dawley rats; SAH, subarachnoid haemorrhage; Figure 1. Experimental design (A–C). SD rats, Sprague–Dawley rats; SAH, subarachnoid haemorrhage; NS, + normal + resveratrol; NS, SAH SAH + normal saline; saline; RES, RES, SAH SAH + resveratrol; Control, Control, SAH SAH ++ control control siRNA; siRNA; siRNA, siRNA, small small interfering interfering RNA; RNA; BBB, BBB, blood-brain blood-brain barrier; barrier; DMSO, DMSO, dimethyl dimethyl sulfoxide; sulfoxide;TXNIP, TXNIP, thioredoxin-interacting thioredoxin-interacting protein; PERK, protein protein kinase kinase RNA-like RNA-like endoplasmic endoplasmic reticulum reticulum (ER) (ER) kinase. kinase. protein; PERK,
First, to detect SAH-induced changes in TXNIP expression and apoptosis, we created SAH models and performed a western blot analysis of the expression of TXNIP, TRX1, p-ASK-1/ASK-1, cleaved caspase-3 (CC3), B-cell lymphoma (BCL)-2-associated X protein (BAX), and BCL-2 at different time intervals (n = 6 in each group). Both CC3 and BAX are common apoptosis indexes, while TRX1 and BCL-2 are anti-apoptotic proteins [9,10]. We found that TXNIP expression increased significantly after SAH (Figure 2A,B) at 24 h (0.31 ± 0.02) in comparison to the sham group (0.15 ±
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First, to detect SAH-induced changes in TXNIP expression and apoptosis, we created SAH models and performed a western blot analysis of the expression of TXNIP, TRX1, p-ASK-1/ASK-1, cleaved caspase-3 (CC3), B-cell lymphoma (BCL)-2-associated X protein (BAX), and BCL-2 at different time intervals (n = 6 in each group). Both CC3 and BAX are common apoptosis indexes, while TRX1 and BCL-2 are anti-apoptotic proteins [9,10]. We found that TXNIP expression increased significantly Int. J. Mol. Sci. 2017, 18, 854 3 of 16 after SAH (Figure 2A,B) at 24 h (0.31 ± 0.02) in comparison to the sham group (0.15 ± 0.05) and peaked 48 h (0.48 ± h0.04, up± to 3.2-fold, p = 0.007). expression of p-ASK-1/ASK-1 (p = 0.014), 0.05) andatpeaked at 48 (0.48 0.04, up to 3.2-fold, p =The 0.007). The expression of p-ASK-1/ASK-1 (p = CC3 (pCC3 = 0.002), and BAX = 0.000) was also to different extents, accompanied by the 0.014), (p = 0.002), and (p BAX (p = 0.000) was elevated also elevated to different extents, accompanied by downregulation of TRX1 (p = 0.023) and BCL-2 (p = 0.008) (Figure 2A,C–G). These results indicated the downregulation of TRX1 (p = 0.023) and BCL-2 (p = 0.008) (Figure 2A,C–G). These results that SAH that significantly promoted promoted the expression of TXNIP and apoptosis proteins. proteins. indicated SAH significantly the expression of TXNIP and apoptosis
Figure expression of TXNIP and apoptosis proteinsproteins at different time points after SAH.after (A) Figure 2.2. TheThe expression of TXNIP and apoptosis at different time points Representative western blot of TXNIP, thioredoxin 1 (TRX1), p-ASK-1/ASK-1, cleaved caspase-3 SAH. (A) Representative western blot of TXNIP, thioredoxin 1 (TRX1), p-ASK-1/ASK-1, (CC3), lymphoma protein (BAX), andX BCL-2; cleavedB-cell caspase-3 (CC3),(BCL)-2-associated B-cell lymphomaX(BCL)-2-associated protein (B–G) (BAX),Densitometric and BCL-2; quantification of the protein band optical densities for TXNIP and associated proteins. TXNIP (B–G) Densitometric quantification of the protein band optical densities for TXNIP and associated expression increased significantly after SAH and was accompanied by an increased expression of proteins. TXNIP expression increased significantly after SAH and was accompanied by an increased apoptosis proteins and decreased expression of anti-apoptotic proteins. # sham vs. SAH at 24 h, p < expression of apoptosis proteins and decreased expression of anti-apoptotic proteins. # sham vs. SAH 0.05. apoptosis kinase-1. kinase-1. at 24 ASK-1, h, p < 0.05. ASK-1,signal-regulating apoptosis signal-regulating
2.2. Colocalized with with TUNEL TUNEL Positive Positive Cells Cells 2.2. TXNIP TXNIP Was Was Expressed Expressed in in Neurons Neurons and and Is Is Colocalized Based on our our previous previousclinical clinicaland andexperimental experimental observations, clinical symptoms appear Based on observations, clinical symptoms appear andand are are significant at 24 h in patients and animals after intracranial aneurysm bleeding and SAH. significant at 24 h in patients and animals after intracranial aneurysm bleeding and SAH. Meanwhile, Meanwhile, in our previous study, we detectedbrain significant brain cellatapoptosis at 24 h and usually in our previous study, we detected significant cell apoptosis 24 h and usually remove the remove the brain tissue at 24 h [3,11]. As such, at the beginning of the experimental design, we brain tissue at 24 h [3,11]. As such, at the beginning of the experimental design, we aimed to observe aimed to observe results at 24 hexperiments, in the following experiments, including immunofluorescence, the results at 24 hthe in the following including immunofluorescence, western blot, and western blot, and apoptosis detection. In addition, we found that changes in TXNIP apoptosis detection. In addition, we found that changes in TXNIP expression began at 12 hexpression and were began at at 1248 h h. and obvious 48 h. At the time, differences expression 24 h obvious Atwere the same time,atdifferences in same TXNIP expression at 24inh TXNIP was also significantatwhen was also with significant when compared the sham group. Therefore,western the results of compared the sham group. Therefore,with the results of immunofluorescence, blot, and immunofluorescence, western blot, and apoptosis detection at 24 h may also provide powerful apoptosis detection at 24 h may also provide powerful evidence to support research conclusions. evidence to support research conclusions. Because a previous study reported TXNIP expression in neurons of ischaemic–reperfusion Because a previous study reported expression neurons ischaemic–reperfusion brains [12], we also investigated the tissue TXNIP expression of TXNIPinafter SAH. of Immunofluorescence was brains [12], we also investigated the tissue expression of TXNIP after SAH. Immunofluorescence was performed to detect the localization of TXNIP in the SAH rat brain. Our results showed that TXNIP performed detect the of of TXNIP in the SAH Our results was widelyto expressed in localization the cytoplasm neurons after SAHrat (nbrain. = 3, Figure 3A). Toshowed confirmthat that TXNIP TXNIP was widely expressed in the cytoplasm of neurons after SAH (n = 3, Figure 3A). To confirm that was associated with brain cell apoptosis after SAH, we used Terminal dexynucleotidyl transferase TXNIP was associated with brain cell apoptosis after SAH, we used Terminal dexynucleotidyl (TdT)-mediated dUTP nick end labeling (TUNEL) staining, a marker for apoptotic cell death [13]. transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining, a marker for apoptotic cell death [13]. Interestingly, TUNEL analysis demonstrated for the first time that TXNIP was widely colocalized with TUNEL-positive cells in both the hippocampus and cortex of SAH rats (n = 3, Figure 3B). These results may provide the critical evidence necessary to support the proapoptotic effect of TXNIP after SAH.
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Interestingly, TUNEL analysis demonstrated for the first time that TXNIP was widely colocalized with TUNEL-positive cells in both the hippocampus and cortex of SAH rats (n = 3, Figure 3B). These results may Int.provide J. Mol. Sci.the 2017,critical 18, 854 evidence necessary to support the proapoptotic effect of TXNIP after 4SAH. of 16
Figure 3. 3. The analysis of of TXNIP TXNIP and and immunofluorescence immunofluorescenceTerminal Terminal Figure The histological histological fluorescent fluorescent analysis dexynucleotidyl (TdT)-mediated dUTP end labeling (TUNEL) staining with dexynucleotidyltransferase transferase (TdT)-mediated dUTP nicknick end labeling (TUNEL) staining with TXNIP. TXNIP. (A) TXNIP was colocalized with NEUN-positive cells (the neurons) (n = 3, detected (A) TXNIP was colocalized with NEUN-positive cells (the neurons) (n = 3, detected by fluorescence by microscopy, fluorescenceFV1200, microscopy, FV1200, Olympus, Japan). NEUN TXNIP (green), (red), DAPI Olympus, Japan). TXNIP (green), (red), DAPINEUN (4′,6-diamidino-2(40 ,6-diamidino-2-phenylindole, the nucleus, blue), and original magnification × 1200; (B) TXNIP phenylindole, the nucleus, blue), and original magnification ×1200; (B) TXNIP was colocalized with was colocalized with TUNEL-positive cells in the hippocampus and subcortex (n = 3, detected TUNEL-positive cells in the hippocampus and subcortex (n = 3, detected by fluorescence microscopy,by fluorescence microscopy, Olympus, (red), TUNEL (theoriginal nucleus, IX71, Olympus, Japan).IX71, TXNIP (red), Japan). TUNELTXNIP (green), DAPI (the (green), nucleus,DAPI blue), blue), original magnification of the subcortex (×200) and hippocampus (×400). bars: = 20 µm, magnification of the subcortex (×200) and hippocampus (×400). Scale bars: (A) =Scale 20 μm, (B)(A) = 120 μm (B)(top) = 120and µm60(top) and 60 µm (bottom). μm (bottom).
Downregulation TXNIPbybyResveratrol Resveratrol(RES) (RES) and and siRNA siRNA 2.3.2.3. Downregulation ofofTXNIP furtherconfirm confirmour our hypothesis hypothesis that induces apoptosis afterafter SAH,SAH, we used ToTo further thatTXNIP TXNIP induces apoptosis we RES usedand RES TXNIP siRNA to downregulate TXNIP expression. RES is reported to have anti-apoptotic, and TXNIP siRNA to downregulate TXNIP expression. RES is reported to have anti-apoptotic, anti-oxidative, and anti-inflammatory properties [14] and is the most commonly reported strategy to anti-oxidative, and anti-inflammatory properties [14] and is the most commonly reported strategy suppress TXNIP and mRNA expression in animal experiments [7,15]. We detected that TXNIP to suppress TXNIP and mRNA expression in animal experiments [7,15]. We detected that TXNIP expression increased at 24 h (0.41 ± 0.01) in comparison to the sham group (Figure 4D,E, 0.08 ± 0.01, expression increased at 24 h (0.41 ± 0.01) in comparison to the sham group (Figure 4D,E, 0.08 ± 0.01, p = 0.001). We found that TXNIP blockage by RES in SAH could significantly reduce the generation p = 0.001). We found that TXNIP blockage by RES in SAH could significantly reduce the generation of apoptosis proteins (Figure 4D). Meanwhile, RES prevented SAH-induced brain injury and was of associated apoptosis with proteins (Figure 4D). Meanwhile, RES prevented SAH-induced brain injury and was the reduced expression of TXNIP (p = 0.01), p-ASK-1 (p = 0.005), CC3 (p = 0.000), and associated with the reduced expression of TXNIP (p =(p0.01), p-ASK-1 (p =(p0.005), CC3 (p h= after 0.000), BAX (p = 0.001) and the increased expression of TRX1 = 0.000) and BCL-2 = 0.001) at 24 and BAX(Figures (p = 0.001) andand the S2). increased of TRX1 = 0.000)byand BCL-2 (p at of 24 h SAH 4D–F Brain expression cell apoptosis was (p analysed counting the= 0.001) number after SAH (Figure 4D–F and Figure S2). Brain cell apoptosis was analysed by counting the number TUNEL-positive cells (n = 5), and the number of TUNEL-positive cells in the cortex was reduced by of TUNEL-positive (n Figure = 5), and the number of TUNEL-positive cells in the cortex was reduced by RES injection (pcells < 0.05, 4G,H). RES injection (p
