- Email: [email protected]
Inhibition of TRIM8 restrains ischaemia-reperfusion-mediated cerebral injury by regulation of NF-κB activation associated inflammation and apoptosis
Journal Pre-proof Inhibition of TRIM8 restrains ischaemia-reperfusion-mediated cerebral injury by regulation of NF-κB activation associated inflammation and apoptosis Xue Bai, Yan-Li Zhang, Li-Ning Liu PII:
S0014-4827(20)30006-9
DOI:
https://doi.org/10.1016/j.yexcr.2020.111818
Reference:
YEXCR 111818
To appear in:
Experimental Cell Research
Received Date: 2 October 2019 Revised Date:
3 December 2019
Accepted Date: 3 January 2020
Please cite this article as: X. Bai, Y.-L. Zhang, L.-N. Liu, Inhibition of TRIM8 restrains ischaemiareperfusion-mediated cerebral injury by regulation of NF-κB activation associated inflammation and apoptosis, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2020.111818. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Authorship contribution statement Xue Bai, Yan-Li Zhang and Li-Ning Liu designed and performed the experiments. Xue Bai and Yan-Li Zhang analyzed the data and drafted the manuscript. Li-Ning Liu provided the fund support.
Graphical Abstract
Inhibition of TRIM8 restrains ischaemia-reperfusion-mediated cerebral injury by regulation of NF-κB activation associated inflammation and apoptosis
Xue Bai #1, Yan-Li Zhang
#2
, Li-Ning Liu *3
1. Department of Neurology, Shanxi Provincial People's Hospital, Taiyuan, Sh anxi 030012, China. 2. Department of Neurology Rehabilitation Ward, Heze Municipal Hospital, S handong Province, Heze 274000, China. 3. Department of Neurology, The second Affiliated Hospital of Xi'an Medical University, Xi'an, shaanxi 710038, China.
* Dr. Li-Ning Liu as the corresponding author: Dr. Li-ning Liu; Department of Neurology, The second Affiliated Hospital of Xi'an Medical Un iversity, Xi'an, shaanxi 710038, China. Email: [email protected] #
The first two authors contributed equally to this work.
Abstract. Stroke is a leading global cause of mortality and disability. However, the pathogenesis that contributes to stroke has not been fully understood. The tripartite motif (TRIM)-containing proteins usually exhibit essential regulatory roles during various biological processes. TRIM8 is a RING domain-containing E3 ubiquitin ligase, playing crucial roles in regulating inflammation and apoptosis. In the present study, we reported that TRIM8 expression was significantly induced in the peri-infarct cortex area of mice after stroke onset. TRIM8 siRNA in vivo transfection resulted in the attenuated cognitive impairments in mice with cerebral ischaemiareperfusion (IR) injury. In addition, TRIM8 knockdown was neuroprotective, as evidenced by the reduced infarct area, decreased neurological deficit score and down-regulated number of TUNEL-positive cells in the peri-infarct area. Moreover, TRIM8 inhibition obviously repressed glial fibrillary acidic protein (GFAP) expression in peri-hematoma cortex and hippocampus. Furthermore, inflammation induced by cerebral IR injury was highly restrained by TRIM8 knockdown in serum, peri-infarct area and hippocampus, which were along with the remarkable decreases in the phosphorylated expression of IκB kinase alpha (IKKα), inhibitory κB α (IκBα) and nuclear factor kappa B (NF-κB). Moreover, TRIM8 knockdown significantly reduced apoptosis in hippocampus of mice with cerebral IR injury by reducing Caspase-3 cleavage. The in vitro experiment confirmed the neuroprotective role of TRIM8-knockdown in regulating cerebral IR injury. Intriguingly, we found that TRIM8 over-expression-promoted inflammatory response and apoptosis could be markedly attenuated by the inactivation of NF-κB signaling through pre-treatment of JSH-23 or QNZ in lipopolysaccharide (LPS)-incubated astrocytes (ASTs). Therefore, TRIM8 positively regulated cerebral IR injury by activating NF-κB pathway to enhance inflammation and apoptosis. Targeting TRIM8 could provide feasible therapeutic treatment for stroke. Keywords: TRIM8, cerebral IR injury, inflammation, apoptosis, NF-κB
1. Introduction Stroke is a major global cause of death and permanent disability [1]. However, few effective therapeutic strategies have been shown to be successful in improving patient outcomes [2,3]. Inflammatory response and apoptosis are essential fundamental mechanisms that significantly promote stroke
progression
via
regulating
various
signaling
pathways
[4,5].
Nevertheless, the underlying molecular mechanisms integrating these pathways are not clear. Tripartite motif 8 (TRIM8) is a ubiquitously expressed factor in the TRIM family that orchestrates a large number of biological processes, such as cell survival, differentiation, apoptosis and, especially, the innate immune response [6,7]. According to previous studies, TRIM8 was suggested to be involved in the development of cancer and immune diseases deriving from their potent meditation on inflammation [8,9]. The human TRIM8 gene maps to chromosome 10q24.3, a region that shows frequent deletion or loss of heterozygosity in glioblastomas [10]. TRIM8 is also designated glioblastomaexpressed RING-finger protein (GERP). In addition, TRIM8 may participate in the carcinogenesis and progression of glioma and the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients [6,11]. TRIM8 has been reported as a regulator of stemness and self-renewal capabilities of glioma stem cells through signal transducer and activator of transcription-3 (STAT3) activation [12]. Thus, TRIM8 might play a critical role in regulating the pathological changes of brain tissues. Given the crucial involvements of inflammation and apoptosis during stroke progression, we supposed that TRIM8 might be of importance for the pathogenesis of stroke; however, the underlying mechanism has yet been investigated. In the study, the in vivo and in vitro experiments were performed to explore the effects of TRIM8 on cerebral IR injury. We found that TRIM8 expression was significantly increased in peri-infarct area of mice with IR injury. TRIM8 knockdown alleviated cognitive impairments induced by cerebral IR injury. In addition, suppressing TRIM8 expression markedly
attenuated
ischaemia-induced
inflammation
and
apoptosis
in
peri-
hematoma cortex and hippocampus of mice. Importantly, the in vitro study suggested that inactivating NF-κB signaling obviously alleviated TRIM8promoted inflammatory response and apoptosis in LPS-incubated ASTs. Thus, our study identified TRIM8 as a novel signaling in cerebral IR injury. 2. Materials and methods 2.1. Animals and treatments All animal procedures were approved by the Animal Care and Use Committee of the Second Hospital of Jilin University (Jilin, China). Animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication revised in 1996). Male, 8-10 weeks old, C57BL/6J mice, weighed 20-22 g, were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animals were housed in individual cages with free access to sterile acidified water and irradiated food in a SPF facility. To knockdown the TRIM8 gene, a specific expression siRNAs against mouse TRIM8 (5’-CCUCUACGUAUATGGCUGUCT-3’; 5’-UAGGCATACTAUGUACGGAGC3’) were purchased from GenScript (Nanjing, Jiangsu Province, China). A scrambled
siRNA
was
used
as
a
negative
control
(5’-
UAACUGUCACCUCCTGUTGGU-3’; 5’-GGCGACGACATTGAUACGUAU-3’). 50 ug of TRIM8 siRNA (siTRIM8) or scrambled siRNA (siRNA) was diluted in 25 uL Entranster-in vivo reagent (Engreen Biosystem Co., Ltd., Beijing, China) and 10% glucose mixture following the manufacturer’s protocols [13]. Each mouse was intravenously injected with the siRNA mix at the final dose of 250 ug/ml in a volume of 200 uL. After 3 d of TRIM8 siRNA in vivo transfection, all mice were subjected to cerebral IR experimental protocols by occluding the left middle cerebral artery (MCA) using the intraluminal filament technique [14]. The mice in which the filament was withdrawn immediately after the decline in regional cerebral blood flow (rCBF) were used as sham controls. After 24 h of reperfusion, animals were sacrificed, and the isolated fresh brain samples were subjected to further studies.
2.2. Astrocytes (AST) incubation AST cells were isolated from the cortex of postnatal day 3 C57BL6 mice as previously described [15]. The isolated AST was incubated in plates and cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco) and 100 KU/L penicillin and 100 mg/L streptomycin at 37°C, 5% CO2. TRIM8 siRNA sequences and negative control (NC) siRNA sequences were obtained from GenScript. The coding region of TRIM8 complementary DNA (cDNA) was cloned into the pcDNA 3.1(+) vector (pcDNA-TRIM8), and the constructs were constructed by Cyagen (Guangzhou, China) and verified by DNA sequencing. AST cells were transfected using Lipofectamine 2000 reagent (Invitrogen, USA) for 24 h. NF-κB inhibitors, including JSH-23 and QNZ, were purchased from Selleck (USA) to repress NF-κB signaling. 2.3. Immunofluorescent staining Animal brains were sectioned at a 20 µm thickness using a cryostat. The sections were then blocked in 5% normal donkey serum diluted in PBS for 1 h at room temperature and then incubated overnight at 4°C with TRIM8 (Santa Cruz, USA) as the primary antibody. Alexa-Fluor 488 was used as secondary fluorescent probe, followed by incubation with DAPI for nuclear staining (Beyotime, Shanghai, China). The sections were viewed by a fluorescent microscopy. 2.4. Real time-quantitative PCR (RT-qPCR) analysis Total RNA was isolated from AST or frozen tissues using TRIZOL reagent (Invitrogen) according to the manufacturer’s protocols, and 2 µg of RNA was reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, USA). Quantitative RT-PCR analysis was carried out with the LightCycler 480 SYBR Green 1 Master Mix (Roche) on the LightCycler 480 QPCR System (Roche). The PCR conditions were 95°C for 10 min; 40 cycles of 95°C for 10 s, 60°C for 10 s and 72°C for 20 s; and a final extension at 72°C for 10 min. GAPDH was served as the internal control. The primer sequences were shown as followings:
TRIM8 forward, 5’-AGTGTCCGTATCCAATCCG-3’; TRIM8 reverse, 5’-CCGTACCCACAACGGACTC-3’; IL-6 forward, 5’-GTATGCGGAGAATACACA-3’; IL-6 reverse, 5’-CGGAATACCCACACCGATCTA-3’; TNF-α forward, 5’-CGGTTGGACCTATTAGAG-3’; TNF-α reverse, 5’-AGCAGGATCTAAGGGAT-3’; IL-1β forward, 5’-TAATAGACGTAGGTGAATCA-3’; IL-1β reverse, 5’-CTCCAATCTGAACAGTA-3’; MCP-1 forward, 5’-AGATGGCATGTTATGTCA-3’; MCP-1 reverse, 5’-CACAACGAGTCGATAGGA-3’; GAPDH forward, 5’-TAAGGTGCAGCATCCAC-3’; GAPDH reverse, 5’-CGTATACAGAGACGCAGAAG-3’. 2.5. Western blotting analysis The isolated peri-hematoma cortex and hippocampus tissues from mice without perfusion, as well as the cell samples were mechanically lysed using ice-cold RIPA lysis buffer (Beyotime). Then, the lysates were centrifuged at 12,000 × g for 15 min at 4◦C, and protein concentration was measured using an enhanced BCA protein assay kit (Beyotime). Next, protein samples (20-50 µg/lane
for
samples)
were
separated
through
SDS-PAGE
and
electrotransferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk for 1.5 h at room temperature followed by incubation with primary antibodies, including antiTRIM8, anti-p-NF-κB, anti-p-IKKα, anti-p-IκBα, anti-Caspase-3 and antiGAPDH. All primary antibodies were purchased from Abcam (USA) and diluted at 1:1000. Then, membranes were incubated with HRP-conjugated secondary antibodies. GAPDH was used as a loading control. Finally, protein
bands were visualized using ECL system (Beyotime). The relative quantity of proteins was analyzed using Image J (Version 1.37, NIH, USA). 2.6. Cognitive determination After MCAO operation for 7 d, the animals were trained and tested in a Morris water maze (MWM) as previously described [16]. Briefly, mice were given 60 s to find the visible platform. Then, the platform was moved to just below the opaque water surface for the place navigation test, and mice performed 4 trials per day, with an interval of 1 min, for 5 consecutive days. For each trial, their spatial learning scores, including the latency period required to escape onto the platform and the path length, were recorded. 2.7. ELISA measurements Commercial kits purchased from Roche System (USA) were used for serum or medium interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and IL-6 determination according to the manufacturer’s protocols. 2.8. 2,3,5-triphenyltetrazolium chloride (TTC) staining and histochemical analysis Neurological deficits were assessed after 24 h of reperfusion using Bederson’s score [17]. After the mice were decapitated, the brains were removed and sliced into 2.0-mm-thick sections. The brain sections were incubated in a 2% TTC solution (Sigma Aldrich, USA) for 30 min at 37°C and then fixed in 4% paraformaldehyde solution. TTC-stained slices were captured using a digital scanner. The infarction area in each section was determined using Image J software. To minimize the error induced by brain edema, total infarct area was manually evaluated according to the following formulas: Infarct volume = contralateral hemisphere region - non-infarcted region in the ipsilateral hemisphere Infarct percentage = infarct v/v of the contralateral hemisphere × 100%. Brain samples were fixed in 4% paraformaldehyde. Serial coronal sections (3 µm thickness) were prepared. The slices were incubated with primary antibody of GFAP (Abcam, dilution
at 1:100) overnight at 4°C after blocking with 5% goat serum. Then, slices were incubated with secondary antibody (Beyotime) at 37°C for 1 h. 3’-3’diaminobenzidine tetrahydrochloride (DAB, Beyotime) was applied to the slides and allowed to react for 5 min. Sections were then stained with hematoxylin, dehydrated in ethanol, and mounted in neutral gum. The number of GFAP-positive cells in cerebral cortex and hippocampus was quantified in six randomly selected fields in one slice. As for Nissl staining, brain sections were immersed in 0.1% cresyl violet at 37°C for 20 min. After washing with distilled water, sections were dehydrated, fitted with coverslips and examined with a light microscope. Neurons with round and palely stained nuclei were served as surviving cells, whereas shrunken cells with pyknotic nuclei were considered as dying cells. 2.9. TUNEL staining According to the manufacturer’s protocol for the in situ Cell Death Detection Kit (Roche, Mannheim, Germany), TUNEL staining was performed to calculate cell death in brain sections. Six random fields from each group were observed with a fluorescence microscope. 2.10. Flow cytometry analysis Apoptosis in AST was examined using a fluorescein isothiocyanate-labeled AnnexinV/PI Apoptosis Detection Kit (KeyGen Biotechology, Nanjing, China) using flow cytometry (FACSCalibur, Becton Dickinson, USA) following the manufacturer’s instructions. 2.11. Statistical analysis Data are expressed as the mean ± SEM. Differences among groups were analyzed using analysis of variance, followed by a post hoc Tukey’s test. Comparisons between two groups were performed by an unpaired Student’s t-test. P < 0.05 was considered statistically significant. 3. Results 3.1. TRIM8 expression is increased after cerebral IR injury
To investigate the role of TRIM8 in regulating cerebral IR injury, an experimental stroke model was induced by MCAO for 1 h followed by various periods of reperfusion. As shown in Fig. 1A-D, TRIM8 expression levels in the peri-infarct area were significantly increased compared to the contralateral control following I/R. In vitro, LPS treatment time- and dose-dependently induced TRIM8 expression in AST (Fig. 1E and F). Therefore, TRIM8 was dramatically promoted in brain tissues of mice following IR or in AST challenged with LPS. 3.2. TRIM8 deficiency alleviates cerebral and cognitive impairments In the hidden platform tests, the mice from siRNA in the IR group showed significantly longer escape latency than the sham control group. As expected, the path lengths were also longer in IR/siRNA group than the Sham/siRNA mice. However, the results of these tests for mice transfected with siTRIM8 were significantly decreased compared with the IR/siRNA group (Fig. 2A-C). TRIM8 knockdown led to significant reduction in infarct volumes compared with those in siRNA mice 24 h after MCAO, along with an obvious decrease in neurological deficit score (Fig. 2D-F). In addition, we also found that TRIM8 knockdown exhibited significant role in alleviating the infarction area of mice 1 h after 60-min of MCAO (Supplementary fig. 1A and B), confirming the regulatory effects of TRIM8 on cerebral IR in mice. As shown in Fig. 2G, TRIM8 inhibition significantly reduced the number of TUNELpositive cells in peri-hematoma cortex after cerebral IR injury compared with the IR/siRNA group. Nissl staining suggested that cerebral IR injury decreased the number of Nissl positive cells in hippocampus, indicating the significant cell death of neuronal cells, which was markedly alleviated by TRIM8 inhibition (Fig. 2H). Caspase-3 is a pivotal effector of apoptosis in ischemic stroke [18]. Moreover, TRIM8 knockdown obviously down-regulated the cleaved Caspase-3 expression in hippocampus of IR mice in comparison to IR/siRNA group (Fig. 2I). Collectively, inhibiting TRIM8 after cerebral IR could be neuroprotective. 3.3. TRIM8 suppression attenuates cerebral IR-induced inflammation and apoptosis
GFAP expression was highly induced by MCAO in peri-hematoma cortex and hippocampus from siRNA mice, whereas being obviously down-regulated in TRIM8-knockdown mice (Fig. 3A and B). ELISA and RT-qPCR analysis suggested remarkable increases of TNF-α, IL-1β, IL-6 and MCP-1 in serum, peri-hematoma cortex and hippocampus isolated from siRNA group of mice. Notably, TRIM8 knockdown markedly reduced the release or expression of these signals after cerebral IR injury (Fig. 3C-E). In addition, LPS significantly promoted the secretion of TNF-α, IL-1β, IL-6 and MCP-1 in the culture medium collected from Con/siRNA AST, which was markedly downregulated by TRIM8 silence (Fig. 3F). Furthermore, we discovered that MCAO clearly elevated the expression of p-IKKα, p-IκBα and p-NF-κB in perihematoma cortex and hippocampus of siRNA mice, while being reversed in TRIM8-knockdown mice (Fig. 3G). Consistently, LPS-induced over-expression of p-IKKα, p-IκBα and p-NF-κB in AST was obviously decreased in AST with the loss of TRIM8 (Fig. 3H). Additionally, the expression of cleaved Caspase3 induced by LPS was apparently inhibited by TRIM8 knockdown, accompanied with a remarkable reduction of apoptosis (Fig. 3I and J). Thus, the results above suggested that TRIM8 could modulate inflammation and apoptosis to meditate cerebral IR injury. 3.4. TRIM8 regulates NF-κB signaling to meditate inflammation and apoptosis in LPS-incubated AST To further explore the effects of TRIM8 oin cerebral IR injury, we overexpressed TRIM8 expression in AST (Fig. 4A). As shown in Fig. 4B, LPSinduced expression of TNF-α, IL-1β, IL-6 and MCP-1 was further promoted by TRIM8 over-expression. Consistently, the expression of p-IKKα, p-IκBα and pNF-κB induced by LPS in AST was significantly enhanced by TRIM8 overexpression (Fig. 4C). In addition, LPS-induced apoptosis was obviously elevated in AST with TRIM8 over-expression, which was along with the enhanced expression of cleaved Caspase-3 (Fig. 4D and E). As shown in Fig. 4F, we found that pcTRIM8-promoted mRNA levels of TNF-α, IL-1β, IL-6 and MCP-1 in LPS-stimulated AST were significantly suppressed by the pretreatment of JSH-23 or QNZ. Also, the activation of IKKα, IκBα and NF-κB
accelerated by TRIM8 over-expression was restrained by JSH-23 or QNZ pretreatment in the LPS-incubated AST (Fig. 4G). In addition, pre-treatment of JSH-23 or QNZ significantly abrogated pcTRIM8-elevated apoptosis in LPSincubated AST (Fig. 4H). Cleavage of Caspase-3 promoted by pcTRIM8 was markedly diminished in LPS-incubated AST with the pre-treatment of JSH-23 or QNZ (Fig. 4I). Together, these in vitro results above indicated that NF-κB signaling played an essential role in TRIM8-regulated stroke. 4. Discussion In spite of extensive studies of the molecular mechanisms of ischemic cerebral injury, little progression has been made in translating findings into clinical practice. At least in part, the short of appropriate therapies for stroke is the insufficient understanding of the pathologies that contribute to stroke. Here, we provided new insights into the pathology of stroke using mice challenged with cerebral IR injury. TRIM8 is a ubiquitously expressed E3 ligase, and is involved in the innate immune response, cell cycle, and cell death [6-9,19]. However, the regulatory effect of TRIM8 on cerebral IR injury remains largely unknown. In the study, for the first time we found that TRIM8 was a critical positive modulator of cerebral IR injury. TRIM8 was over-expressed
in
peri-hematoma
cortex
after
cerebral
IR
injury.
Suppressing TRIM8 markedly attenuated cerebral IR injury-induced cognitive impairments. TRIM8 knockdown in vivo exhibited neuroprotective effects, as proved by the reduced infarct area and neurological deficit score. Further investigation into the underlying molecular mechanisms suggested that TRIM8 knockdown alleviated inflammation and apoptosis in peri-hematoma cortex and/or hippocampus of mice with cerebral IR injury, which was largely regulated by the activation of NF-κB signaling. Inflammation and apoptosis are characteristic features/complications of stroke, which could mutually promote each other to form a vicious circle [4,5,20,21]. Increasing evidence indicated that post-ischemic inflammation results in the progression of neuronal injury and cerebral infarction. In inflammatory response, endogenous mediators, such as cytokine and chemokine, are involved. Pro-inflammatory cytokines play a critical role in
the attraction of leukocytes as potential inducers of chemokine. Thereby, cytokines function as essential regulators to attract leucocytes in the condition of inflammation [22,23]. Moreover, activated astrocytes could produce cytokines and
chemokine,
which are
responsible for
the
accumulation of inflammatory cells in the damaged brain tissue. TNF-α, IL1β, IL-6 and MCP-1 are the potential factors initiating inflammatory reactions [24]. The ischemic brain was detected with promoted expression of TNF-α, IL-6, MCP-1 and IL-1β, which are considered as a part of tissue damage in IR injury [25]. NF-κB signaling has been firmly confirmed to accelerate the development of cerebral IR injury by promoting the release of inflammatory cytokines [26]. A positive role of TRIM8 in inflammation activation has been reported, partly through the modulation of NF-κB pathway
[7,27].
In
our
study,
MCAO-induced
AST
activation
and
inflammation was attenuated by TRIM8 knockdown, as evidenced by the reduction of GFAP, TNF-α, IL-1β, IL-6 and MCP-1 in peri-hematoma cortex and hippocampus of mice, which was largely through reducing the expression of p-IKKα, p-IκBα and p-NF-κB. These observations indicated that TRIM8 knockdown might be a suppressor of inflammation and NF-κB signaling in response to cerebral IR injury. Apoptosis is another fundamental mechanism that occurs during ischemic brain injury [5,28]. Furthermore, upon ischemic stress, brain tissues exhibit biological hallmarks of apoptosis, including the effector of Caspase-3 [29]. In addition, TRIM8 was suggested to interact with p53 to enhance apoptosis, preventing tumor growth [30,31]. Therefore, TRIM8 may have a prominent role in apoptosis induction. In our study, active Caspase-3 was highly induced by MCAO, which was consistent with the intensity of apoptosis. However, the enhancement of apoptosis was clearly suppressed by TRIM8 knockdown. NF-κB, as a crucial transcription factor, plays an essential role in inducing apoptosis [32,33]. Here, we found that TRIM8-accelerated inflammation and apoptosis in LPS-incubated AST were abrogated by the pre-treatment of LSH-23 or QNZ, which are essential NF-κB inhibitors. Thus, we hypothesized that TRIM8-regulated inflammatory response and apoptosis was largely dependent on the activation of NF-κB signaling.
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Figure legends Figure 1. TRIM8 expression is increased after cerebral IR injury. Timecourse of TRIM8 mRNA levels (A) and protein expression (B) in perihematoma cortex after cerebral IR injury. n = 8 in each group. (C,D) Immunofluorescence study of TRIM8-positive levels in peri-hematoma cortex after cerebral IR injury. n = 5 in each group. (E) AST was treated with 100 ng/ml LPS for 0, 6, 12 or 24 h. Then, cells were harvested for RT-qPCR and western blot analysis of TRIM8. n = 4 in each group. (F) Various concentrations of LPS were subjected to AST for 24 h, followed by RT-qPCR and western blot analysis of TRIM8. n = 4 in each group. All data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 and
***
p < 0.001 versus Con group
without any treatments. Figure 2. TRIM8 deficiency alleviates cerebral and cognitive impairments. (A) Representative images of the path chart of animals in MWM were showed. n = 8 in each group. (B) The escape latency of mice at the indicated time after cerebral IR injury. n = 8 in each group. (C) Path length of mice at the indicated time after cerebral IR injury. n = 8 in each group. (D) Brains stained with TTC after IR. n = 8 in each group. Quantification of (E) infarction area and (F) neurological deficit score. n = 8 in each group. (G) TUNEL staining of peri-hematoma cortex after cerebral IR injury. n = 6 in each group. (H) Nissl staining of CA1 area in mice after IR injury. n = 6 in each group. (I) Western blot analysis of cleaved Caspase-3 in hippocampus of mice after IR injury. n = 5 in each group. All data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 and 0.05 and
***
p < 0.001 versus Sham/siRNA group; #p <
##
p < 0.01 versus IR/siRNA group.
Figure 3. TRIM8 suppression attenuates cerebral IR-induced inflammation and apoptosis. Immunohistochemical analysis of GFAP in (A) peri-hematoma cortex and (B) CA1 area of hippocampus from mice after cerebral IR injury. n = 6 in each group. (C) ELISA analysis of TNF-α, IL-1β, IL-6 and MCP-1 in serum of each group of mice. n = 8 in each group. RT-qPCR analysis of TNF-α, IL-1β, IL-6 and MCP-1 in (D) peri-hematoma cortex and (E) hippocampus of mice after cerebral IR injury. n = 5 in each group. (F) ELISA analysis of TNF-
α, IL-1β, IL-6 and MCP-1 in medium of AST after LPS (100 ng/ml) treatment for 24 h. n = 5 in each group. (G) Western blot analysis of p-IKKα, p-IκBα and p-NF-κB in peri-hematoma cortex and hippocampus of mice. n = 5 in each group. Western blot analysis of (H) p-IKKα, p-IκBα and p-NF-κB, as well as (I) cleaved Caspase-3 in 100 ng/ml of LPS-exposed AST for 24 h. n = 5 in each group. (J) Flow cytometry analysis of apoptosis in AST treated with 24 h of LPS (100 ng/ml). n = 5 in each group. All data are shown as the mean ± SEM. **
p < 0.01 and
##
p < 0.01 and
***
p < 0.001 versus Sham/siRNA or Con/siRNA group; #p < 0.05,
###
p < 0.001 versus IR/siRNA or LPS/siRNA group.
Figure 4. TRIM8 regulates NF-κB signaling to meditate inflammation and apoptosis in LPS-incubated AST. (A) AST was transfected with pcDNA or TRIM8 pcDNA to over-express TRIM8. After 24 h of transfection, all cells were harvested for western blot analysis of TRIM8. n = 5 in each group. AST was transfected with TRIM8 pcDNA for 24 h, followed by 100 ng/ml of LPS treatment for another 24 h. Then, all cells were collected for the subsequent studies. (B) RT-qPCR analysis of TNF-α, IL-1β, IL-6 and MCP-1 in AST treated as showed. n = 4 in each group. (C) Western blot analysis of pIKKα, p-IκBα and p-NF-κB in AST treated as described. n = 5 in each group. (D) Flow cytometry analysis of cellular apoptosis. n = 5 in each group. (E) Western blot analysis of cleaved Caspase-3 in AST treated as indicated. n = 5 in each group. Next, AST was pre-treated with the inhibitors of NF-κB (JSH23 and QNZ) at the indicated concentrations for 2 h, followed by transfection with pcDNA/TRIM8 for 24 h. Then, all cells were exposed to LPS (100 ng/ml) for another 24 h. (F) RT-qPCR analysis of TNF-α, IL-1β, IL-6 and MCP-1 in AST treated as indicated. n = 4 in each group. (G) Western blot analysis of p-IKKα, p-IκBα and p-NF-κB in AST. n = 5 in each group. (H) Flow cytometry analysis of apoptosis in AST treated as described. n = 5 in each group. (I) Western blot analysis of cleaved Caspase-3 in cells. n = 5 in each group. (J) Proposed molecular mechanism of TRIM8-regulated IR cerebral injury. All data are shown as the mean ± SEM.
***
p < 0.001 versus Con/pcDNA
group; #p < 0.05 versus LPS/pcDNA group; +p < 0.05 and
++
p < 0.01.
Figure 1-revision
Figure 2
Figure 3
Figure 4
Highlights
TRIM8 deficiency alleviates cerebral and cognitive impairments TRIM8 suppression attenuates cerebral IR-induced inflammation and apoptosis TRIM8 regulates NF-κB signaling to meditate inflammation and apoptosis in LPS-incubated AST
Conflict of interest The authors see no any conflicts of interest in this work.
S0014-4827(20)30006-9
DOI:
https://doi.org/10.1016/j.yexcr.2020.111818
Reference:
YEXCR 111818
To appear in:
Experimental Cell Research
Received Date: 2 October 2019 Revised Date:
3 December 2019
Accepted Date: 3 January 2020
Please cite this article as: X. Bai, Y.-L. Zhang, L.-N. Liu, Inhibition of TRIM8 restrains ischaemiareperfusion-mediated cerebral injury by regulation of NF-κB activation associated inflammation and apoptosis, Experimental Cell Research (2020), doi: https://doi.org/10.1016/j.yexcr.2020.111818. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Authorship contribution statement Xue Bai, Yan-Li Zhang and Li-Ning Liu designed and performed the experiments. Xue Bai and Yan-Li Zhang analyzed the data and drafted the manuscript. Li-Ning Liu provided the fund support.
Graphical Abstract
Inhibition of TRIM8 restrains ischaemia-reperfusion-mediated cerebral injury by regulation of NF-κB activation associated inflammation and apoptosis
Xue Bai #1, Yan-Li Zhang
#2
, Li-Ning Liu *3
1. Department of Neurology, Shanxi Provincial People's Hospital, Taiyuan, Sh anxi 030012, China. 2. Department of Neurology Rehabilitation Ward, Heze Municipal Hospital, S handong Province, Heze 274000, China. 3. Department of Neurology, The second Affiliated Hospital of Xi'an Medical University, Xi'an, shaanxi 710038, China.
* Dr. Li-Ning Liu as the corresponding author: Dr. Li-ning Liu; Department of Neurology, The second Affiliated Hospital of Xi'an Medical Un iversity, Xi'an, shaanxi 710038, China. Email: [email protected] #
The first two authors contributed equally to this work.
Abstract. Stroke is a leading global cause of mortality and disability. However, the pathogenesis that contributes to stroke has not been fully understood. The tripartite motif (TRIM)-containing proteins usually exhibit essential regulatory roles during various biological processes. TRIM8 is a RING domain-containing E3 ubiquitin ligase, playing crucial roles in regulating inflammation and apoptosis. In the present study, we reported that TRIM8 expression was significantly induced in the peri-infarct cortex area of mice after stroke onset. TRIM8 siRNA in vivo transfection resulted in the attenuated cognitive impairments in mice with cerebral ischaemiareperfusion (IR) injury. In addition, TRIM8 knockdown was neuroprotective, as evidenced by the reduced infarct area, decreased neurological deficit score and down-regulated number of TUNEL-positive cells in the peri-infarct area. Moreover, TRIM8 inhibition obviously repressed glial fibrillary acidic protein (GFAP) expression in peri-hematoma cortex and hippocampus. Furthermore, inflammation induced by cerebral IR injury was highly restrained by TRIM8 knockdown in serum, peri-infarct area and hippocampus, which were along with the remarkable decreases in the phosphorylated expression of IκB kinase alpha (IKKα), inhibitory κB α (IκBα) and nuclear factor kappa B (NF-κB). Moreover, TRIM8 knockdown significantly reduced apoptosis in hippocampus of mice with cerebral IR injury by reducing Caspase-3 cleavage. The in vitro experiment confirmed the neuroprotective role of TRIM8-knockdown in regulating cerebral IR injury. Intriguingly, we found that TRIM8 over-expression-promoted inflammatory response and apoptosis could be markedly attenuated by the inactivation of NF-κB signaling through pre-treatment of JSH-23 or QNZ in lipopolysaccharide (LPS)-incubated astrocytes (ASTs). Therefore, TRIM8 positively regulated cerebral IR injury by activating NF-κB pathway to enhance inflammation and apoptosis. Targeting TRIM8 could provide feasible therapeutic treatment for stroke. Keywords: TRIM8, cerebral IR injury, inflammation, apoptosis, NF-κB
1. Introduction Stroke is a major global cause of death and permanent disability [1]. However, few effective therapeutic strategies have been shown to be successful in improving patient outcomes [2,3]. Inflammatory response and apoptosis are essential fundamental mechanisms that significantly promote stroke
progression
via
regulating
various
signaling
pathways
[4,5].
Nevertheless, the underlying molecular mechanisms integrating these pathways are not clear. Tripartite motif 8 (TRIM8) is a ubiquitously expressed factor in the TRIM family that orchestrates a large number of biological processes, such as cell survival, differentiation, apoptosis and, especially, the innate immune response [6,7]. According to previous studies, TRIM8 was suggested to be involved in the development of cancer and immune diseases deriving from their potent meditation on inflammation [8,9]. The human TRIM8 gene maps to chromosome 10q24.3, a region that shows frequent deletion or loss of heterozygosity in glioblastomas [10]. TRIM8 is also designated glioblastomaexpressed RING-finger protein (GERP). In addition, TRIM8 may participate in the carcinogenesis and progression of glioma and the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients [6,11]. TRIM8 has been reported as a regulator of stemness and self-renewal capabilities of glioma stem cells through signal transducer and activator of transcription-3 (STAT3) activation [12]. Thus, TRIM8 might play a critical role in regulating the pathological changes of brain tissues. Given the crucial involvements of inflammation and apoptosis during stroke progression, we supposed that TRIM8 might be of importance for the pathogenesis of stroke; however, the underlying mechanism has yet been investigated. In the study, the in vivo and in vitro experiments were performed to explore the effects of TRIM8 on cerebral IR injury. We found that TRIM8 expression was significantly increased in peri-infarct area of mice with IR injury. TRIM8 knockdown alleviated cognitive impairments induced by cerebral IR injury. In addition, suppressing TRIM8 expression markedly
attenuated
ischaemia-induced
inflammation
and
apoptosis
in
peri-
hematoma cortex and hippocampus of mice. Importantly, the in vitro study suggested that inactivating NF-κB signaling obviously alleviated TRIM8promoted inflammatory response and apoptosis in LPS-incubated ASTs. Thus, our study identified TRIM8 as a novel signaling in cerebral IR injury. 2. Materials and methods 2.1. Animals and treatments All animal procedures were approved by the Animal Care and Use Committee of the Second Hospital of Jilin University (Jilin, China). Animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication revised in 1996). Male, 8-10 weeks old, C57BL/6J mice, weighed 20-22 g, were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animals were housed in individual cages with free access to sterile acidified water and irradiated food in a SPF facility. To knockdown the TRIM8 gene, a specific expression siRNAs against mouse TRIM8 (5’-CCUCUACGUAUATGGCUGUCT-3’; 5’-UAGGCATACTAUGUACGGAGC3’) were purchased from GenScript (Nanjing, Jiangsu Province, China). A scrambled
siRNA
was
used
as
a
negative
control
(5’-
UAACUGUCACCUCCTGUTGGU-3’; 5’-GGCGACGACATTGAUACGUAU-3’). 50 ug of TRIM8 siRNA (siTRIM8) or scrambled siRNA (siRNA) was diluted in 25 uL Entranster-in vivo reagent (Engreen Biosystem Co., Ltd., Beijing, China) and 10% glucose mixture following the manufacturer’s protocols [13]. Each mouse was intravenously injected with the siRNA mix at the final dose of 250 ug/ml in a volume of 200 uL. After 3 d of TRIM8 siRNA in vivo transfection, all mice were subjected to cerebral IR experimental protocols by occluding the left middle cerebral artery (MCA) using the intraluminal filament technique [14]. The mice in which the filament was withdrawn immediately after the decline in regional cerebral blood flow (rCBF) were used as sham controls. After 24 h of reperfusion, animals were sacrificed, and the isolated fresh brain samples were subjected to further studies.
2.2. Astrocytes (AST) incubation AST cells were isolated from the cortex of postnatal day 3 C57BL6 mice as previously described [15]. The isolated AST was incubated in plates and cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco) and 100 KU/L penicillin and 100 mg/L streptomycin at 37°C, 5% CO2. TRIM8 siRNA sequences and negative control (NC) siRNA sequences were obtained from GenScript. The coding region of TRIM8 complementary DNA (cDNA) was cloned into the pcDNA 3.1(+) vector (pcDNA-TRIM8), and the constructs were constructed by Cyagen (Guangzhou, China) and verified by DNA sequencing. AST cells were transfected using Lipofectamine 2000 reagent (Invitrogen, USA) for 24 h. NF-κB inhibitors, including JSH-23 and QNZ, were purchased from Selleck (USA) to repress NF-κB signaling. 2.3. Immunofluorescent staining Animal brains were sectioned at a 20 µm thickness using a cryostat. The sections were then blocked in 5% normal donkey serum diluted in PBS for 1 h at room temperature and then incubated overnight at 4°C with TRIM8 (Santa Cruz, USA) as the primary antibody. Alexa-Fluor 488 was used as secondary fluorescent probe, followed by incubation with DAPI for nuclear staining (Beyotime, Shanghai, China). The sections were viewed by a fluorescent microscopy. 2.4. Real time-quantitative PCR (RT-qPCR) analysis Total RNA was isolated from AST or frozen tissues using TRIZOL reagent (Invitrogen) according to the manufacturer’s protocols, and 2 µg of RNA was reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, USA). Quantitative RT-PCR analysis was carried out with the LightCycler 480 SYBR Green 1 Master Mix (Roche) on the LightCycler 480 QPCR System (Roche). The PCR conditions were 95°C for 10 min; 40 cycles of 95°C for 10 s, 60°C for 10 s and 72°C for 20 s; and a final extension at 72°C for 10 min. GAPDH was served as the internal control. The primer sequences were shown as followings:
TRIM8 forward, 5’-AGTGTCCGTATCCAATCCG-3’; TRIM8 reverse, 5’-CCGTACCCACAACGGACTC-3’; IL-6 forward, 5’-GTATGCGGAGAATACACA-3’; IL-6 reverse, 5’-CGGAATACCCACACCGATCTA-3’; TNF-α forward, 5’-CGGTTGGACCTATTAGAG-3’; TNF-α reverse, 5’-AGCAGGATCTAAGGGAT-3’; IL-1β forward, 5’-TAATAGACGTAGGTGAATCA-3’; IL-1β reverse, 5’-CTCCAATCTGAACAGTA-3’; MCP-1 forward, 5’-AGATGGCATGTTATGTCA-3’; MCP-1 reverse, 5’-CACAACGAGTCGATAGGA-3’; GAPDH forward, 5’-TAAGGTGCAGCATCCAC-3’; GAPDH reverse, 5’-CGTATACAGAGACGCAGAAG-3’. 2.5. Western blotting analysis The isolated peri-hematoma cortex and hippocampus tissues from mice without perfusion, as well as the cell samples were mechanically lysed using ice-cold RIPA lysis buffer (Beyotime). Then, the lysates were centrifuged at 12,000 × g for 15 min at 4◦C, and protein concentration was measured using an enhanced BCA protein assay kit (Beyotime). Next, protein samples (20-50 µg/lane
for
samples)
were
separated
through
SDS-PAGE
and
electrotransferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk for 1.5 h at room temperature followed by incubation with primary antibodies, including antiTRIM8, anti-p-NF-κB, anti-p-IKKα, anti-p-IκBα, anti-Caspase-3 and antiGAPDH. All primary antibodies were purchased from Abcam (USA) and diluted at 1:1000. Then, membranes were incubated with HRP-conjugated secondary antibodies. GAPDH was used as a loading control. Finally, protein
bands were visualized using ECL system (Beyotime). The relative quantity of proteins was analyzed using Image J (Version 1.37, NIH, USA). 2.6. Cognitive determination After MCAO operation for 7 d, the animals were trained and tested in a Morris water maze (MWM) as previously described [16]. Briefly, mice were given 60 s to find the visible platform. Then, the platform was moved to just below the opaque water surface for the place navigation test, and mice performed 4 trials per day, with an interval of 1 min, for 5 consecutive days. For each trial, their spatial learning scores, including the latency period required to escape onto the platform and the path length, were recorded. 2.7. ELISA measurements Commercial kits purchased from Roche System (USA) were used for serum or medium interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and IL-6 determination according to the manufacturer’s protocols. 2.8. 2,3,5-triphenyltetrazolium chloride (TTC) staining and histochemical analysis Neurological deficits were assessed after 24 h of reperfusion using Bederson’s score [17]. After the mice were decapitated, the brains were removed and sliced into 2.0-mm-thick sections. The brain sections were incubated in a 2% TTC solution (Sigma Aldrich, USA) for 30 min at 37°C and then fixed in 4% paraformaldehyde solution. TTC-stained slices were captured using a digital scanner. The infarction area in each section was determined using Image J software. To minimize the error induced by brain edema, total infarct area was manually evaluated according to the following formulas: Infarct volume = contralateral hemisphere region - non-infarcted region in the ipsilateral hemisphere Infarct percentage = infarct v/v of the contralateral hemisphere × 100%. Brain samples were fixed in 4% paraformaldehyde. Serial coronal sections (3 µm thickness) were prepared. The slices were incubated with primary antibody of GFAP (Abcam, dilution
at 1:100) overnight at 4°C after blocking with 5% goat serum. Then, slices were incubated with secondary antibody (Beyotime) at 37°C for 1 h. 3’-3’diaminobenzidine tetrahydrochloride (DAB, Beyotime) was applied to the slides and allowed to react for 5 min. Sections were then stained with hematoxylin, dehydrated in ethanol, and mounted in neutral gum. The number of GFAP-positive cells in cerebral cortex and hippocampus was quantified in six randomly selected fields in one slice. As for Nissl staining, brain sections were immersed in 0.1% cresyl violet at 37°C for 20 min. After washing with distilled water, sections were dehydrated, fitted with coverslips and examined with a light microscope. Neurons with round and palely stained nuclei were served as surviving cells, whereas shrunken cells with pyknotic nuclei were considered as dying cells. 2.9. TUNEL staining According to the manufacturer’s protocol for the in situ Cell Death Detection Kit (Roche, Mannheim, Germany), TUNEL staining was performed to calculate cell death in brain sections. Six random fields from each group were observed with a fluorescence microscope. 2.10. Flow cytometry analysis Apoptosis in AST was examined using a fluorescein isothiocyanate-labeled AnnexinV/PI Apoptosis Detection Kit (KeyGen Biotechology, Nanjing, China) using flow cytometry (FACSCalibur, Becton Dickinson, USA) following the manufacturer’s instructions. 2.11. Statistical analysis Data are expressed as the mean ± SEM. Differences among groups were analyzed using analysis of variance, followed by a post hoc Tukey’s test. Comparisons between two groups were performed by an unpaired Student’s t-test. P < 0.05 was considered statistically significant. 3. Results 3.1. TRIM8 expression is increased after cerebral IR injury
To investigate the role of TRIM8 in regulating cerebral IR injury, an experimental stroke model was induced by MCAO for 1 h followed by various periods of reperfusion. As shown in Fig. 1A-D, TRIM8 expression levels in the peri-infarct area were significantly increased compared to the contralateral control following I/R. In vitro, LPS treatment time- and dose-dependently induced TRIM8 expression in AST (Fig. 1E and F). Therefore, TRIM8 was dramatically promoted in brain tissues of mice following IR or in AST challenged with LPS. 3.2. TRIM8 deficiency alleviates cerebral and cognitive impairments In the hidden platform tests, the mice from siRNA in the IR group showed significantly longer escape latency than the sham control group. As expected, the path lengths were also longer in IR/siRNA group than the Sham/siRNA mice. However, the results of these tests for mice transfected with siTRIM8 were significantly decreased compared with the IR/siRNA group (Fig. 2A-C). TRIM8 knockdown led to significant reduction in infarct volumes compared with those in siRNA mice 24 h after MCAO, along with an obvious decrease in neurological deficit score (Fig. 2D-F). In addition, we also found that TRIM8 knockdown exhibited significant role in alleviating the infarction area of mice 1 h after 60-min of MCAO (Supplementary fig. 1A and B), confirming the regulatory effects of TRIM8 on cerebral IR in mice. As shown in Fig. 2G, TRIM8 inhibition significantly reduced the number of TUNELpositive cells in peri-hematoma cortex after cerebral IR injury compared with the IR/siRNA group. Nissl staining suggested that cerebral IR injury decreased the number of Nissl positive cells in hippocampus, indicating the significant cell death of neuronal cells, which was markedly alleviated by TRIM8 inhibition (Fig. 2H). Caspase-3 is a pivotal effector of apoptosis in ischemic stroke [18]. Moreover, TRIM8 knockdown obviously down-regulated the cleaved Caspase-3 expression in hippocampus of IR mice in comparison to IR/siRNA group (Fig. 2I). Collectively, inhibiting TRIM8 after cerebral IR could be neuroprotective. 3.3. TRIM8 suppression attenuates cerebral IR-induced inflammation and apoptosis
GFAP expression was highly induced by MCAO in peri-hematoma cortex and hippocampus from siRNA mice, whereas being obviously down-regulated in TRIM8-knockdown mice (Fig. 3A and B). ELISA and RT-qPCR analysis suggested remarkable increases of TNF-α, IL-1β, IL-6 and MCP-1 in serum, peri-hematoma cortex and hippocampus isolated from siRNA group of mice. Notably, TRIM8 knockdown markedly reduced the release or expression of these signals after cerebral IR injury (Fig. 3C-E). In addition, LPS significantly promoted the secretion of TNF-α, IL-1β, IL-6 and MCP-1 in the culture medium collected from Con/siRNA AST, which was markedly downregulated by TRIM8 silence (Fig. 3F). Furthermore, we discovered that MCAO clearly elevated the expression of p-IKKα, p-IκBα and p-NF-κB in perihematoma cortex and hippocampus of siRNA mice, while being reversed in TRIM8-knockdown mice (Fig. 3G). Consistently, LPS-induced over-expression of p-IKKα, p-IκBα and p-NF-κB in AST was obviously decreased in AST with the loss of TRIM8 (Fig. 3H). Additionally, the expression of cleaved Caspase3 induced by LPS was apparently inhibited by TRIM8 knockdown, accompanied with a remarkable reduction of apoptosis (Fig. 3I and J). Thus, the results above suggested that TRIM8 could modulate inflammation and apoptosis to meditate cerebral IR injury. 3.4. TRIM8 regulates NF-κB signaling to meditate inflammation and apoptosis in LPS-incubated AST To further explore the effects of TRIM8 oin cerebral IR injury, we overexpressed TRIM8 expression in AST (Fig. 4A). As shown in Fig. 4B, LPSinduced expression of TNF-α, IL-1β, IL-6 and MCP-1 was further promoted by TRIM8 over-expression. Consistently, the expression of p-IKKα, p-IκBα and pNF-κB induced by LPS in AST was significantly enhanced by TRIM8 overexpression (Fig. 4C). In addition, LPS-induced apoptosis was obviously elevated in AST with TRIM8 over-expression, which was along with the enhanced expression of cleaved Caspase-3 (Fig. 4D and E). As shown in Fig. 4F, we found that pcTRIM8-promoted mRNA levels of TNF-α, IL-1β, IL-6 and MCP-1 in LPS-stimulated AST were significantly suppressed by the pretreatment of JSH-23 or QNZ. Also, the activation of IKKα, IκBα and NF-κB
accelerated by TRIM8 over-expression was restrained by JSH-23 or QNZ pretreatment in the LPS-incubated AST (Fig. 4G). In addition, pre-treatment of JSH-23 or QNZ significantly abrogated pcTRIM8-elevated apoptosis in LPSincubated AST (Fig. 4H). Cleavage of Caspase-3 promoted by pcTRIM8 was markedly diminished in LPS-incubated AST with the pre-treatment of JSH-23 or QNZ (Fig. 4I). Together, these in vitro results above indicated that NF-κB signaling played an essential role in TRIM8-regulated stroke. 4. Discussion In spite of extensive studies of the molecular mechanisms of ischemic cerebral injury, little progression has been made in translating findings into clinical practice. At least in part, the short of appropriate therapies for stroke is the insufficient understanding of the pathologies that contribute to stroke. Here, we provided new insights into the pathology of stroke using mice challenged with cerebral IR injury. TRIM8 is a ubiquitously expressed E3 ligase, and is involved in the innate immune response, cell cycle, and cell death [6-9,19]. However, the regulatory effect of TRIM8 on cerebral IR injury remains largely unknown. In the study, for the first time we found that TRIM8 was a critical positive modulator of cerebral IR injury. TRIM8 was over-expressed
in
peri-hematoma
cortex
after
cerebral
IR
injury.
Suppressing TRIM8 markedly attenuated cerebral IR injury-induced cognitive impairments. TRIM8 knockdown in vivo exhibited neuroprotective effects, as proved by the reduced infarct area and neurological deficit score. Further investigation into the underlying molecular mechanisms suggested that TRIM8 knockdown alleviated inflammation and apoptosis in peri-hematoma cortex and/or hippocampus of mice with cerebral IR injury, which was largely regulated by the activation of NF-κB signaling. Inflammation and apoptosis are characteristic features/complications of stroke, which could mutually promote each other to form a vicious circle [4,5,20,21]. Increasing evidence indicated that post-ischemic inflammation results in the progression of neuronal injury and cerebral infarction. In inflammatory response, endogenous mediators, such as cytokine and chemokine, are involved. Pro-inflammatory cytokines play a critical role in
the attraction of leukocytes as potential inducers of chemokine. Thereby, cytokines function as essential regulators to attract leucocytes in the condition of inflammation [22,23]. Moreover, activated astrocytes could produce cytokines and
chemokine,
which are
responsible for
the
accumulation of inflammatory cells in the damaged brain tissue. TNF-α, IL1β, IL-6 and MCP-1 are the potential factors initiating inflammatory reactions [24]. The ischemic brain was detected with promoted expression of TNF-α, IL-6, MCP-1 and IL-1β, which are considered as a part of tissue damage in IR injury [25]. NF-κB signaling has been firmly confirmed to accelerate the development of cerebral IR injury by promoting the release of inflammatory cytokines [26]. A positive role of TRIM8 in inflammation activation has been reported, partly through the modulation of NF-κB pathway
[7,27].
In
our
study,
MCAO-induced
AST
activation
and
inflammation was attenuated by TRIM8 knockdown, as evidenced by the reduction of GFAP, TNF-α, IL-1β, IL-6 and MCP-1 in peri-hematoma cortex and hippocampus of mice, which was largely through reducing the expression of p-IKKα, p-IκBα and p-NF-κB. These observations indicated that TRIM8 knockdown might be a suppressor of inflammation and NF-κB signaling in response to cerebral IR injury. Apoptosis is another fundamental mechanism that occurs during ischemic brain injury [5,28]. Furthermore, upon ischemic stress, brain tissues exhibit biological hallmarks of apoptosis, including the effector of Caspase-3 [29]. In addition, TRIM8 was suggested to interact with p53 to enhance apoptosis, preventing tumor growth [30,31]. Therefore, TRIM8 may have a prominent role in apoptosis induction. In our study, active Caspase-3 was highly induced by MCAO, which was consistent with the intensity of apoptosis. However, the enhancement of apoptosis was clearly suppressed by TRIM8 knockdown. NF-κB, as a crucial transcription factor, plays an essential role in inducing apoptosis [32,33]. Here, we found that TRIM8-accelerated inflammation and apoptosis in LPS-incubated AST were abrogated by the pre-treatment of LSH-23 or QNZ, which are essential NF-κB inhibitors. Thus, we hypothesized that TRIM8-regulated inflammatory response and apoptosis was largely dependent on the activation of NF-κB signaling.
In summary, we have identified TRIM8 as a novel positive meditator in cerebral IR injury. TRIM8 knockdown attenuated MCAO-induced cognitive disorder. Inflammation and apoptosis triggered by cerebral IR injury were alleviated by the suppression of TRIM8, which was largely through the inactivation of NF-κB signaling pathway (Fig. 4J). Thus, our study provided experimental evidence that TRIM8 inhibition might be a novel therapeutic target to develop effective treatments against clinical stroke progression in future. References [1] Gladstone D J, Dorian P, Spring M, et al. Atrial premature beats predict atrial fibrillation in cryptogenic stroke: results from the EMBRACE trial. Stroke, 2015, 46(4): 936-941. [2] Bang O Y, Goyal M, Liebeskind D S. Collateral circulation in ischemic stroke: assessment tools and therapeutic strategies. Stroke, 2015, 46(11): 3302-3309. [3] Gilhus N E, Verschuuren J J. Myasthenia gravis: subgroup classification and therapeutic strategies. The Lancet Neurology, 2015, 14(10): 10231036. [4] Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. Journal of leukocyte biology, 2010, 87(5): 779-789. [5] Lv H, Wang L, Shen J, et al. Salvianolic acid B attenuates apoptosis and inflammation via SIRT1 activation in experimental stroke rats. Brain research bulletin, 2015, 115: 30-36. [6] Micale L, Fusco C, Fontana A, et al. TRIM8 downregulation in glioma affects cell proliferation and it is associated with patients survival. BMC cancer, 2015, 15(1): 470. [7] Ye W, Hu M M, Lei C Q, et al. TRIM8 Negatively Regulates TLR3/4Mediated Innate Immune Response by Blocking TRIF–TBK1 Interaction. The Journal of Immunology, 2017: ji1601647.
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Figure legends Figure 1. TRIM8 expression is increased after cerebral IR injury. Timecourse of TRIM8 mRNA levels (A) and protein expression (B) in perihematoma cortex after cerebral IR injury. n = 8 in each group. (C,D) Immunofluorescence study of TRIM8-positive levels in peri-hematoma cortex after cerebral IR injury. n = 5 in each group. (E) AST was treated with 100 ng/ml LPS for 0, 6, 12 or 24 h. Then, cells were harvested for RT-qPCR and western blot analysis of TRIM8. n = 4 in each group. (F) Various concentrations of LPS were subjected to AST for 24 h, followed by RT-qPCR and western blot analysis of TRIM8. n = 4 in each group. All data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 and
***
p < 0.001 versus Con group
without any treatments. Figure 2. TRIM8 deficiency alleviates cerebral and cognitive impairments. (A) Representative images of the path chart of animals in MWM were showed. n = 8 in each group. (B) The escape latency of mice at the indicated time after cerebral IR injury. n = 8 in each group. (C) Path length of mice at the indicated time after cerebral IR injury. n = 8 in each group. (D) Brains stained with TTC after IR. n = 8 in each group. Quantification of (E) infarction area and (F) neurological deficit score. n = 8 in each group. (G) TUNEL staining of peri-hematoma cortex after cerebral IR injury. n = 6 in each group. (H) Nissl staining of CA1 area in mice after IR injury. n = 6 in each group. (I) Western blot analysis of cleaved Caspase-3 in hippocampus of mice after IR injury. n = 5 in each group. All data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 and 0.05 and
***
p < 0.001 versus Sham/siRNA group; #p <
##
p < 0.01 versus IR/siRNA group.
Figure 3. TRIM8 suppression attenuates cerebral IR-induced inflammation and apoptosis. Immunohistochemical analysis of GFAP in (A) peri-hematoma cortex and (B) CA1 area of hippocampus from mice after cerebral IR injury. n = 6 in each group. (C) ELISA analysis of TNF-α, IL-1β, IL-6 and MCP-1 in serum of each group of mice. n = 8 in each group. RT-qPCR analysis of TNF-α, IL-1β, IL-6 and MCP-1 in (D) peri-hematoma cortex and (E) hippocampus of mice after cerebral IR injury. n = 5 in each group. (F) ELISA analysis of TNF-
α, IL-1β, IL-6 and MCP-1 in medium of AST after LPS (100 ng/ml) treatment for 24 h. n = 5 in each group. (G) Western blot analysis of p-IKKα, p-IκBα and p-NF-κB in peri-hematoma cortex and hippocampus of mice. n = 5 in each group. Western blot analysis of (H) p-IKKα, p-IκBα and p-NF-κB, as well as (I) cleaved Caspase-3 in 100 ng/ml of LPS-exposed AST for 24 h. n = 5 in each group. (J) Flow cytometry analysis of apoptosis in AST treated with 24 h of LPS (100 ng/ml). n = 5 in each group. All data are shown as the mean ± SEM. **
p < 0.01 and
##
p < 0.01 and
***
p < 0.001 versus Sham/siRNA or Con/siRNA group; #p < 0.05,
###
p < 0.001 versus IR/siRNA or LPS/siRNA group.
Figure 4. TRIM8 regulates NF-κB signaling to meditate inflammation and apoptosis in LPS-incubated AST. (A) AST was transfected with pcDNA or TRIM8 pcDNA to over-express TRIM8. After 24 h of transfection, all cells were harvested for western blot analysis of TRIM8. n = 5 in each group. AST was transfected with TRIM8 pcDNA for 24 h, followed by 100 ng/ml of LPS treatment for another 24 h. Then, all cells were collected for the subsequent studies. (B) RT-qPCR analysis of TNF-α, IL-1β, IL-6 and MCP-1 in AST treated as showed. n = 4 in each group. (C) Western blot analysis of pIKKα, p-IκBα and p-NF-κB in AST treated as described. n = 5 in each group. (D) Flow cytometry analysis of cellular apoptosis. n = 5 in each group. (E) Western blot analysis of cleaved Caspase-3 in AST treated as indicated. n = 5 in each group. Next, AST was pre-treated with the inhibitors of NF-κB (JSH23 and QNZ) at the indicated concentrations for 2 h, followed by transfection with pcDNA/TRIM8 for 24 h. Then, all cells were exposed to LPS (100 ng/ml) for another 24 h. (F) RT-qPCR analysis of TNF-α, IL-1β, IL-6 and MCP-1 in AST treated as indicated. n = 4 in each group. (G) Western blot analysis of p-IKKα, p-IκBα and p-NF-κB in AST. n = 5 in each group. (H) Flow cytometry analysis of apoptosis in AST treated as described. n = 5 in each group. (I) Western blot analysis of cleaved Caspase-3 in cells. n = 5 in each group. (J) Proposed molecular mechanism of TRIM8-regulated IR cerebral injury. All data are shown as the mean ± SEM.
***
p < 0.001 versus Con/pcDNA
group; #p < 0.05 versus LPS/pcDNA group; +p < 0.05 and
++
p < 0.01.
Figure 1-revision
Figure 2
Figure 3
Figure 4
Highlights
TRIM8 deficiency alleviates cerebral and cognitive impairments TRIM8 suppression attenuates cerebral IR-induced inflammation and apoptosis TRIM8 regulates NF-κB signaling to meditate inflammation and apoptosis in LPS-incubated AST
Conflict of interest The authors see no any conflicts of interest in this work.