- Email: [email protected]
PRDX1 enhances cerebral ischemia-reperfusion injury through activation of TLR4-regulated inflammation and apoptosis
Biochemical and Biophysical Research Communications 519 (2019) 453e461
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
PRDX1 enhances cerebral ischemia-reperfusion injury through activation of TLR4-regulated inflammation and apoptosis Qiang Liu a, Yuan Zhang b, * a b
Department of Neurology, Yan'an University Affiliated Hospital, Yan'an, Shannxi, 716000, China Department of EMG Evoked Potential Chamber, Heze Municipal Hospital, Shandong Province, Heze City, Shandong Province, 274000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2019 Accepted 13 August 2019 Available online 13 September 2019
Stroke is still a leading cause of death across the world. Despite various signals or molecules that contribute to the pathophysiological process have been investigated, the exact molecular mechanisms revealing stroke damage still remain to be explored. Peroxiredoxin 1 (PRDX1) has been identified as a stress-induced macrophage redox protein with multiple functions. Although PRDX1 is a critical factor related to the regulation of immunity, inflammation, apoptosis and oxidative stress, its effects on cerebral ischemia-reperfusion (I-R) injury were presently unclear. In the study, by using a mouse model of I-R injury, we found that PRDX1 expression was up-regulated during I-R injury in a time-dependent manner. Additionally, PRDX1-knockout mice showed reduced infarction area and alleviated neuropathological scores with decreased brain water contents. Furthermore, cell death and inflammatory response in mice with cerebral I-R injury were markedly attenuated by PRDX1 knockout, which were associated with the blockage of Caspase-3 and nuclear factor-kB (NF-kB) signaling pathways. Mechanistically, PRDX1regulated cerebral I-R injury was through the promotion of toll-like receptor-4 (TLR4), as proved by the evidence that TLR4 suppression abrogated the exacerbated effect of TLR4 on inflammatory response and apoptosis in oxygen and glucose deprivation (OGD)-treated primary microglial cells. These data demonstrated that PRDX1 contributed to cerebral stroke by interacting with TLR4, providing an effective therapeutic approach for cerebral I-R injury. © 2019 Published by Elsevier Inc.
Keywords: PRDX1 Cerebral ischemia-reperfusion injury Apoptosis Inflammation TLR4
1. Introduction Stroke is a major cause leading to death worldwide although advancements have been made in therapeutic approaches, and ischemic stroke accounts for approximate 85% of total stroke [1]. Excessive experimental and clinical studies have indicated that multiple pathophysiological processes are involved in cerebral I-R injury, such as immune response, inflammation, apoptotic cell death, endoplasmic reticulum stress and oxidative stress [2e4]. However, therapeutic strategies based on these molecular mechanisms showed unsatisfied outcomes [5]. Therefore, further studies are still warranted to investigate underlying mechanisms for the treatment of cerebral I-R damage. Peroxiredoxins (PRDXs), as important antioxidant proteins, constitute the potent defense system to sustain redox balance through converting hydrogen peroxide to water [6]. PRDX1 belongs
* Corresponding author. E-mail address: [email protected] (Y. Zhang). https://doi.org/10.1016/j.bbrc.2019.08.077 0006-291X/© 2019 Published by Elsevier Inc.
to PRDXs family, and has been reported as a 23-kDa stress-induced macrophage redox protein with a variety of functions [7,8]. Extracellular PRDX1 plays an essential role in regulating inflammatory response [9]. PRDX1 can function as a molecular chaperone with the ability to control the actions of multiple molecules or as a modulator for transcription [10]. PRDX1 expression during the early phase of extracorporeal membrane oxygenation (ECMO) support in cardiogenic shock patients is involved in the progression of systemic inflammatory response syndrome, accompanied with poor clinical outcomes [11]. Recently, PRDX1 was suggested to be a novel damage-associated molecular pattern, contributing to the development of acute liver injury [12]. Moreover, PRDX1 was involved in the progression of myocardial ischemia reperfusion injury by regulating cell apoptosis [13]. Although these initial clues have been elucidated, the exact effects of PRDX1 on cerebral I-R injury remain unclear. In this study, we found that the expression levels of PRDX1 were significantly up-regulated in cerebral I-R injury mouse model, and in microglial cells treated with OGD. PRDX1 knockout mice displayed alleviated infarction area and neurological scores with
454
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
2. Materials and methods
incubated with the primary antibodies overnight at 4 C, including PRDX1 (ab41906, dilution at 1:150, Abcam, USA), Iba1 (ab178847, dilution at 1:150, Abcam) and TLR4 (ab22048, dilution at 1:100, Abcam). After washing, sections were incubated with appropriate secondary antibodies (Abcam), followed by nuclei staining with DAPI (Sigma Aldrich). TUNEL (Terminal Dexynucleotidyl Transferase(Tdt)-Mediated Dutp Nick End Labeling) apoptosis assay kit was used to determine cell death following the manufacturer's protocol (Beyotime).
2.1. Animals and cerebral I-R injury
2.5. In vitro model for cerebral I-R
All animal procedures were performed according to the institutional guidelines for the Care and Use of Laboratory Animals of Yan'an University Affiliated Hospital (Shan'xi, China). All animal experiments were conducted on 10-12 week-old male mice. PRDX1 knock-out (KO) mice were purchased from Cyagen Biotech (Suzhou, China). A cerebral I-R model was established according to transient blockage of the proximal middle cerebral artery as previously indicated [14]. Briefly, mice were anesthetized using 3% isoflurane. After surgical exposure of the left carotid artery, a 6-0 siliconcoated monofilament was then inserted into the internal carotid artery through the external carotid artery, temporarily blocking blood flow to the middle cerebral artery (MCA). Following blockage for 45 min, the monofilament was carefully retracted from the internal carotid artery. Mice were kept immobilized for 10 min. Throughout the surgery, left cerebral blood flow was monitored using a Doppler flowmeter (Perimed, Sweden). For sham mice, the monofilament was withdrawn immediately after the initial onset of reduced cerebral blood flow. Rectal temperature was kept at 37 ± 0.5 C throughout the operation. All animals were allowed to recover in a 37 C incubator for different time periods with free access to food and water.
Primary microglia were isolated from WT and PRDX1-KO mice on postnatal days 0e2 as previously described by the published protocols [17]. Prior to treatment, microglia from mice were plated in chamber slides (100,000 cells/well) and allowed to adhere for 1 h in serum-free and glutamate-free DMEM/F12 nutrient mixture (Gibco, USA). To mimic I-R conditions in vitro, the cells were exposed to transient OGD for 60 min and then returned to normal culture conditions for different periods [18]. Short interfering RNA (siRNA) and plasmids to over-express PRDX1 or TLR4 were transfected into microglial cells using the Lipofectamine 2000 reagent (Invitrogen, USA) following the manufacturer's instructions. siRNAS against TLR4 and PRDX1, and PRDX1 or TLR4 specific plasmid were synthesized by RiboBio (Guangzhou, China).
decreased brain water contents. Additionally, cerebral I-R injuryinduced apoptosis and inflammatory response were also attenuated in mice with PRDX1 deletion. Furthermore, we elucidated that PRDX1 could interact with TLR4 to regulate NF-kB and cell death, resulting in cerebral I-R damage. Thus, targeting PRDX1/TLR4 represented a potential and effective therapeutic approach against ischemic stroke.
2.2. Quantitative real-time PCR Total RNA was extracted from the ipsilateral striatum of mice using Tripure Isolation Reagent (Roche, USA) according to the manufacturer's instructions for RT-qPCR analysis following standard protocols as previously described [15]. Primer pairs for PCR were presented in Supplement Table 1. 2.3. Western blot analysis Proteins extracted from the ipsilateral striatum of mice using RIPA lysis buffer (Beyotime, Shanghai, China) with protease inhibitor cocktail tablets. The protein expression levels were then measured using primary antibodies (Supplement Table 2) as previously described [16]. Protein expression levels were normalized to GAPDH. 2.4. Immunofluorescence and TUNEL staining For histopathological analysis, animals were euthanized and transcardially perfused with PBS (pH 7.4), followed by 4% paraformaldehyde. Then, brains were slowly removed and immersed in 4% paraformaldehyde for 2 h. Next, the brains were dehydrated using 20% sucrose solution in PBS overnight, followed by 30% sucrose for another 6 h. Subsequently, brains were embedded in Optimal Cutting Temperature Compound. 8-mm-thickness brain sections were made with a cryostat microtome. For antigen retrieval, brain slides were incubated in EDTA at 86 C for 5 min. For primary microglial cells, coverslips were fixed with 4% paraformaldehyde and then incubated in 10% goat serum (Sigma Aldrich, USA). For immunofluorescence labeling, all slides were
2.6. Fluoro-Jade B staining Fluoro-Jade B (Millipore) was used for cell death analysis in brain sections according to the manufacturer's protocols. 2.7. Analysis of infarct size The brains were removed for infract volume calculation through the 2,3,5-triphenyltetrazolium chloride (TTC, Sigma Aldrich) staining. Brain sections were stained in 2% TTC for 30 min at 37 C, and fixed in 10% formaldehyde neutral buffer overnight. The infract tissues were unstained, and the red regions was served as the normal area. 2.8. Neurological impairment score After reperfusion for 24 h, neurological impairment resulted from the tMCAO procedure was calculated according to the ninepoint scale [19]. The investigators were blinded to all measurements of the neurological deficits. 2.9. Brain water content analysis Brains were removed to determine wet weight immediately. Then, the brain samples were dried in an oven at 110 C for 48 h to measure the dry weight. Brain water content was determined as (wet weight-dry weight)/wet weight100%. 2.10. Evans blue extravasation To determine the blood-brain barrier (BBB) permeability at 24 h post-surgery, mice were treated with 100 ml of 4% Evans blue dye (Sigma Aldrich) via retro-orbital injection. After 2 h, we perfused the mouse transcardially with saline, removed the brain. 2.11. Flow cytometry analysis Apoptosis was calculated by fluorescein isothiocyanate (FITC)conjugated Annexin V and propidium iodide (PI) staining
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
455
Fig. 1. PRDX1 expression levels were induced after experiencing cerebral I-R injury in vivo and in vitro. (A) RT-qPCR and (B) western blotting analysis demonstrating the expression levels of PRDX1 in mice after cerebral I-R injury at the indicated time. (C) Immunofluorescent staining of PRDX1 in striatum of mice after cerebral I-R injury. (D) RT-qPCR, (E) western blotting and (F) immunofluorescent analysis indicating the expression levels of PRDX1 in primary isolated microglial cells treated with OGD at the indicated time points. (G) Representative western blot of PRDX1 levels in brain tissues of WT mice and PRDX1-KO mice. (H) Representative images of TTC staining of brain sections from both WT and PRDX1-KO groups after reperfusion for 24 h. Quantitative analysis of (I) infarct volume and (J) neurological scores of the mice from the indicated groups. (K) Representative images of Evans blue extravasation in peri-infarction area at 24 h after cerebral I-R injury. (L) Quantification of Evans blue leakage in mice after cerebral I-R injury for 24 h. (M) Brain water content was examined at 24 h after cerebral I-R injury. Data were expressed as mean ± SEM (n ¼ 8). *P < 0.05, **P < 0.01 and ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
456
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
Fig. 2. PRDX1 knockout attenuated ischemia-reperfusion injury. (A) Fluoro-Jade B and TUNEL staining of brain sections in mice. Quantification of Fluoro Jade B- and TUNELpositive cells were exhibited. (B) Western blot analysis of Bcl-2, Bax, Bid and cleaved Caspase-3 expression levels in the ipsilateral striatum of mice. (C) TUNEL staining of primary microglial cells treated with OGD for 24 h. TUNEL-positive cells were quantified. (D,E) Flow cytometry analysis of primary microglial cells exposed to 24 h of OGD. Apoptosis rate was analyzed. (F) Western blot analysis for Bcl-2 and Caspase-3 cleavage in primary microglial cells treated with OGD for 24 h. Data were expressed as mean ± SEM (n ¼ 8). * P < 0.05, **P < 0.01 and ***P < 0.001.
(Beyotime) according to the manufacturer's instructions. 2.12. Immunoprecipitation (IP) and GST pull-down Human PRDX1 cDNA was cloned into psi-Flag-C1 and pcDNA5HA-C1 to express Flag-PRDX1, HA-PRDX1 and GST-HA-PRDX1 fusion protein, respectively. TLR4-expressing vectors were created
by the same method. IP analysis of PRDX1 and TLR4 were conducted to calculate the interaction of each protein under normal and OGD conditions as previously described [16,18]. GST Pull-down analysis was conducted to determine the direct binding between purified PRDX1 and TLR4 proteins also as previously indicated [16,18].
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
457
Fig. 3. PRDX1 knockout alleviated inflammatory response after ischemia-reperfusion injury. RT-qPCR results for (A) Iba1, (B) TNF-a, COX2, IL-1b, IL-6 and IL-10 in the ipsilateral striatum of mice. (C) Western blot analysis for p-IkBa and p-NF-kB in the ipsilateral striatum of mice. (D) Immunofluorescent staining of Iba-1 and TLR-4 in brain sections of mice from the indicated groups. (E) Western blot analysis for TLR4 and MyD88 in the ipsilateral striatum of mice. (F) Western blot analysis for TLR4, MyD88, p-IkBa and p-NF-kB in primary microglial cells treated with OGD for 24 h. Data were expressed as mean ± SEM (n ¼ 8). *P < 0.05, **P < 0.01 and ***P < 0.001.
2.13. Statistical analysis
3. Results
Data were expressed as mean ± SEM using Graph Pad Prism 6.0 software. One-way analysis of variance (ANOVA) was performed followed by Bonferroni's post-hoc analysis between multiple groups. Comparisons between two groups were performed by Student's t-test. Value of p < 0.05 was considered statistically significant.
3.1. PRDX1 expression levels were enhanced after experiencing cerebral I-R injury in vivo and in vitro In this part, we first found that PRDX1 expression from mRNA and protein levels detected by RT-qPCR and western blotting were markedly increased in a time-dependent manner after cerebral I-R
458
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
Fig. 4. PRDX1-induced ischemia-reperfusion injury via TLR4 activation. (A) IP with an HA antibody or control was performed for western blot using a Flag antibody (up panel) or IP with a Flag antibody for western blot with an HA antibody (down panel). (B) GST pull-down analysis in which either GST-tagged PRDX1 (HA-GST-PRDX1) or control HA-GST was used to pull down Flag-TLR4 (left panel) or GST-tagged TLR4 (HA-GST-TLR4) or HA-GST was used to pull down Flag-PRDX1 (right panel). (C) Primary microglial cells were
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
injury (Fig. 1A and B). Immunofluorescent staining suggested that cerebral I-R led to a significant increase in PRDX1-positive microglia in striatum of mice (Fig. 1C). In vitro, the isolated primary microglia treated with OGD showed higher expression levels of PRDX1 also in a time-dependent fashion (Fig. 1D and E). Similar expression change of PRDX1 was observed in OGD-incubated primary microglial cells using immunofluorescent staining (Fig. 1F). Then, to further investigate if PRDX1 played a critical role in modulating cerebral I-R injury, PRDX1-KO mice were used, and identified by western blot analysis (Fig. 1G). After MCAO, PRDX1-KO mice showed remarkable reductions in infarction area and neurological scores (Fig. 1HeJ). BBB permeability at 24 h after MCAO was then assessed using Evans blue extravasation. As shown in Fig. 1K and L, Evans blue leakage was markedly decreased in MCAO mice with PRDX1 deletion. Finally, MCAO-enhanced brain water contents were highly decreased by PRDX1 knockout (Fig. 1M). Together, the findings above strongly demonstrated a critical role for PRDX1 in regulating stroke damage. 3.2. PRDX1 knockout attenuated ischemia-reperfusion injury Fluorescent staining suggested that PRDX1-KO mice showed a sharp decrease in Fluoro Jade B- and TUNEL-positive cells after MCAO operation in comparison with the WT mice (Fig. 2A). In PRDX1-KO mice after MCAO for 24 h, up-regulation in the levels of the pro-apoptotic proteins, including Bax, Bid and cleaved Caspase3, were lower than that in the WT mice. In contrast, expression of the anti-apoptotic factor Bcl2 was higher than that in the WT group of mice following I-R injury (Fig. 2B). In vitro, primary microglial cells subjected to OGD also showed significant downregulation of TUNEL-positive cells with PRDX1 deficiency (Fig. 2C). Flow cytometry analysis also demonstrated that OGDinduced apoptosis in primary microglia was greatly alleviated by PRDX1 ablation (Fig. 2D and E). Results by western blotting suggested that anti-apoptotic signal Bcl-2 expression levels were markedly rescued by PRDX1 knockout in OGD-stimulated microglial cells. However, opposite results were detected in the expression change of cleaved Caspase-3 (Fig. 2F). 3.3. PRDX1 knockout alleviated inflammatory response after ischemia-reperfusion injury Microglial activation and inflammation play critical roles in the development of cerebral I-R injury [20,21]. Here, the mRNA levels of Iba1, a critical microglial marker, were significantly induced by MCAO, while being reduced in the PRDX1-KOgroup (Fig. 3A). Then, we found that the mRNA levels for pro-inflammatory regulators, such as TNF-a, COX2, IL-1b and IL-6, were markedly reduced in the PRDX1-KO group after MCAO operation, while anti-inflammatory modulator IL-10 mRNA levels were restored (Fig. 3B). Moreover, western blotting demonstrated that the phosphorylation of IkBa and NF-kB (p65) was clearly reduced in PRDX1-KO mice (Fig. 3C). TLR4 is of great importance in regulating inflammatory response via NF-kB signaling [22]. In addition, PRDX1 was reported to meditate TLR4 signaling under various conditions [23]. Then, immunofluorescent staining demonstrated that TLR4 expression levels stimulated by MCAO were markedly alleviated by PRDX1
459
deletion (Fig. 3D). Consistently, PRDX1-KO mice exhibited significantly reduced the expression levels of TLR4 and its downstreaming signal MyD88 following MCAO (Fig. 3E). As expected, TLR4, MyD88, phosphorylation levels of IkBa and NF-kB stimulated by OGD in primary microglial cells were greatly attenuated by the PRDX1 deletion (Fig. 3F). Altogether, these results indicated that, following MCAO, PRDX1 contributed to the pathological inflammation that resulted in cerebral I-R injury. 3.4. PRDX1-induced ischemia-reperfusion injury directly dependent on TLR4 activation We further investigated the interaction of PRDX1 and TLR4 to explore the molecular mechanism of TLR4 suppression by PRDX1 deletion in cerebral I/R injury. HA-tagged PRDX1 and Flag-tagged TLR4 were overexpressed in 293T cells. IP analysis indicated that PRDX1 co-immunoprecipitated with TLR4 and vice versa regardless of the OGD insult (Fig. 4A). A GST-tagged PRDX1 effectively pulled down TLR4, and GST-tagged TLR4 could pull down PRDX1 (Fig. 4B). Thus, we supposed that PRDX1 could directly interact with TLR4. To further reveal if PRDX1-induced cerebral injury was TLR4dependent, primary microglial cells with PRDX1 and TLR4 knockdown and/or over-expression were generated (Fig. 4C). When microglial cells were exposed to OGD, however, co-transfection with siTLR4 counteracted oePRDX1-induced expression of proinflammatory cytokines TNF-a, COX2, IL-1b and IL-6 (Fig. 4D) and apoptosis (Fig. 4E). Conversely, siPRDX1-reduced inflammation and apoptosis were clearly abolished by TLR4 over-expression in OGDexposed microglial cells (Fig. 4F and G). PRDX1-induced cerebral I-R injury was largely TLR4-dependent. 4. Discussion Ischemic stroke is considered as a leading global cause for mortality and disability [1e3]. Despite multiple pathological processes have been targeted to improve ischemic brain injury, effective translation of these findings into clinical treatment still require further studies to reveal the underlying molecular mechanisms. In the present study, PRDX1 might be an up-streaming modulator to regulate inflammation and apoptosis after MCAO. Importantly, PRDX1/TLR4 axis showed a potential role in controlling cerebral I-R damage (Fig. 4H). The most significant finding of our present study is that PRDX1 could interact with TKR4 to regulate NF-kB and apoptosis, leading to cerebral I-R injury. PRDX1 is a small protein (23 kDa) in the Peroxiredoxins family, which is composed of ubiquitously expressed enzymes that down-regulate peroxide levels [5,6]. PRDX family proteins are key initiators of post-ischemic inflammation in the brain [24]. PRDX1 is well-known for its protective effects on different diseases including aging, neurodegenerative diseases and cancers due to its anti-oxidative capability [25]. However, a significant increase of PRDX1 in serum of patients with non-small cell lung cancer was discovered [26]. Moreover, intracellular PRDX1 is released to the extracellular space when respond to stimuli such as TGF-b1, LPS and TNF-a [27]. Recently, a significant promotion of circulating PRDX1 in mice with acute liver injury was discovered, which is associated with the release of pro-
transfected with PRDX1 siRNA, PRDX1 plasmid, TLR4 siRNA or TLR4 plasmid to inhibit or promote PRDX1 and TLR4 expressions, respectively, for 24 h. Then, all cells were harvested for transfection efficacy calculation using western blot analysis. (D,E) Primary microglial cells were transfected with siTLR4, oePRDX1 or the two in combination for 24 h. Then, these cells were subjected to OGD treatment for another 24 h. Next, (D) RT-qPCR analysis was used to determine TNF-a, COX2, IL-1b, IL-6 and IL-10 mRNA levels in cells. (E) Flow cytometry analysis was used to measure apoptosis. (F,G) Primary microglia were transfected with oeTLR4, siPRDX1 or the two in combination for 24 h. Next, the cells were treated with OGD for another 24 h. Subsequently, (F) TNF-a, COX2, IL-1b, IL-6 and IL-10 mRNA levels in cells were assessed using RT-qPCR analysis. (G) Flow cytometry analysis was conducted to evaluate apoptosis. (H) Diagram depicting the interaction between PRDX1 and TLR4 that resulted in cerebral I-R injury. Data were expressed as mean ± SEM (n ¼ 4). * P < 0.05, **P < 0.01 and ***P < 0.001.
460
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
inflammatory cytokines such as IL-1b, IL-6 and TNF-a through activating NF-kB signaling; however, mice with PRDX1 deficiency were protected from acute liver injury and its associated inflammatory response [12]. Inflammatory response plays an important role after cerebral ischemia. Brain damage results in activation of resident microglia after ischemia, which is followed by infiltration of immunocytes, such as neutrophils, T cells, and macrophages [28]. A variety of pro-inflammatory regulators are induced including TNF-a, COX2, IL-1b and IL-6, accelerating brain damage. Suppressing microglia activation could represent an effective therapeutic target to alleviate acute cerebral ischemia [29]. Here, in the present study, we first found that PRDX1 expression in microglial cells was significantly induced by cerebral I-R injury both in vivo and in vitro. Then, PRDX1-KO mice exhibited alleviated cerebral I-R injury induced by MCAO, which was largely associated with the suppression of pro-inflammatory cytokines (TNF-a, COX2, IL-1b and IL-6) and improvement of anti-inflammatory factor (IL10). Similar inhibitory effects of PRDX1 deletion on inflammatory response were confirmed in OGD-treated primary microglial cells, and these effects were attributed to the inactivation of NF-kB signaling. Apoptosis plays a critical role in the pathophysiology of cerebral hypoperfusion. Bcl-2, Bax and Bid are representatively proteins; Caspase-3 is another crucial factor that contributes to the progression of apoptosis [30]. Bcl-2 is important for anti-apoptosis, while Bax and Bis are key pro-apoptotic signals. The ratio of Bcl2/Bax is proposed to determine the apoptotic fate of the cell [31]. In this work, we confirmed that MCAO led to apoptosis in the injured brain samples, as evidenced by the reduced expression levels of Bcl-2, and enhanced levels of Bax, Bid and subsequent Caspase-3 cleavage. Notably, this pro-apoptotic process was greatly blunted by PRDX1 knockout, attenuating cerebral I-R injury. PRDX1 may contribute to apoptosis inhibition in hepatic cancer cells under normal non-stressed conditions [32]. Thus, PRDX1-regulated cerebral I-R injury was also associated with its regulation to apoptotic condition. TLR4 is a key PRR, which binds and contacts down-streaming NF-kB through receptor dimerization. Activated components of the NF-kB signaling pathway meditate inflammation by promoting the expression of TNF-a, COX2, IL-1b and IL-6, as well as other proinflammatory cytokines or chemokine [33]. Suppressing the activation of the TLR4 signaling is a potential anti-inflammatory treatment and may prevent ischemic stroke [34]. Unexpectedly, extracellular PRDX1 has recently been reported as a novel damageassociated molecular pattern (DAMP) attributed to its proinflammatory property through binding to TLR4 [35]. In this study, we not only elucidated that PRDX1 regulated TLR4 expression and the subsequent inflammatory cytokine production during the ischemic I-R injury, but also demonstrated that the promoting effects of PRDX1 on the activation NF-kB signaling and apoptosis depended on the interaction and activation of TLR4. These data provided a therapeutic insight into the PRDX1/TLR4 regulatory axis during the progression of cerebral I-R injury. In summary, findings in this work illustrated a novel function of PRDX1 in regulating ischemic stroke through interacting with TLR4, which subsequently promoted inflammatory response and apoptosis through NF-kB and Caspase-3, respectively (Fig. 4H). Herein, targeting PRDX1 could be insightful for developing new treatments against ischemic stroke for patients.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.077.
References [1] V.L. Feigin, B. Norrving, G.A. Mensah, Global burden of stroke, Circ. Res. 120 (3) (2017) 439e448. [2] T.C. Burns, G.K. Steinberg, Stem cells and stroke: opportunities, challenges and strategies, Expert Opin. Biol. Ther. 11 (4) (2011) 447e461. [3] A.K. Saenger, R.H. Christenson, Stroke biomarkers: progress and challenges for diagnosis, prognosis, differentiation, and treatment, Clin. Chem. 56 (1) (2010) 21e33. [4] T.C. Burns, G.K. Steinberg, Stem cells and stroke: opportunities, challenges and strategies, Expert Opin. Biol. Ther. 11 (4) (2011) 447e461. [5] M.A. Yenari, T.M. Kauppinen, R.A. Swanson, Microglial activation in stroke: therapeutic targets, Neurotherapeutics 7 (4) (2010) 378e391. [6] Y. Zhou, S. Duan, Y. Zhou, et al., Sulfiredoxin-1 attenuates oxidative stress via Nrf2/ARE pathway and 2-Cys Prdxs after oxygen-glucose deprivation in astrocytes, J. Mol. Neurosci. 55 (4) (2015) 941e950. [7] B. Turner-Ivey, Y. Manevich, J. Schulte, et al., Role for Prdx1 as a specific sensor in redox-regulated senescence in breast cancer, Oncogene 32 (45) (2013) 5302. ant, C. Che ry, A. Oussalah, et al., A PRDX1 mutant allele causes a [8] J.L. Gue MMACHC secondary epimutation in cblC patients, Nat. Commun. 9 (1) (2018) 67. [9] D. Liu, P. Mao, Y. Huang, et al., Proteomic analysis of lung tissue in a rat acute lung injury model: identification of PRDX1 as a promoter of inflammation, Mediat. Inflamm. 2014 (4) (2014), 469358. [10] J. Cao, J. Schulte, A. Knight, et al., Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity, EMBO J. 28 (10) (2009) 1505e1517. [11] C.H. Liu, S.W. Kuo, L.M. Hsu, et al., Peroxiredoxin 1 induces inflammatory cytokine response and predicts outcome of cardiogenic shock patients necessitating extracorporeal membrane oxygenation: an observational cohort study and translational approach, J. Transl. Med. 14 (1) (2016) 114. [12] Y. He, S. Li, D. Tang, et al., Circulating Peroxiredoxin-1 is a novel damageassociated molecular pattern and aggravates acute liver injury via promoting inflammation, Free Radic. Biol. Med. 137 (2019) 24e36. [13] W. Guo, X. Liu, J. Li, et al., Prdx1 alleviates cardiomyocyte apoptosis through ROS-activated MAPK pathway during myocardial ischemia/reperfusion injury, Int. J. Biol. Macromol. 112 (2018) 608e615. [14] T. Li, J.J. Qin, X. Yang, et al., The ubiquitin E3 ligase TRAF6 exacerbates ischemic stroke by ubiquitinating and activating Rac1, J. Neurosci. 37 (50) (2017) 12123e12140. [15] L. Wang, L. Wang, Z. Dai, et al., Lack of mitochondrial ferritin aggravated neurological deficits via enhancing oxidative stress in a traumatic brain injury murine model, Biosci. Rep. 37 (6) (2017). BSR20170942. [16] S. Wang, Z.Z. Yan, X. Yang, et al., Hepatocyte DUSP14 maintains metabolic homeostasis and suppresses inflammation in the liver, Hepatology 67 (4) (2018) 1320e1338. [17] J.M. Crain, M. Nikodemova, J.J. Watters, Microglia express distinct M1 and M2 phenotypic markers in the postnatal and adult central nervous system in male and female mice, J. Neurosci. Res. 91 (9) (2013) 1143e1151. [18] Y.Y. Lu, Z.Z. Li, D.S. Jiang, et al., TRAF1 is a critical regulator of cerebral ischaemiaereperfusion injury and neuronal death, Nat. Commun. 4 (2013) 2852. [19] C.F. Xia, R.S. Smith Jr., B. Shen, et al., Postischemic brain injury is exacerbated in mice lacking the kinin B2 receptor, Hypertension 47 (4) (2006) 752e761. [20] M. Lv, Y. Liu, J. Zhang, et al., Roles of inflammation response in microglia cell through Toll-like receptors 2/interleukin-23/interleukin-17 pathway in cerebral ischemia/reperfusion injury, Neuroscience 176 (2011) 162e172. [21] M.D. Bianco-Batlles, A. Sosunov, R.A. Polin, et al., Systemic inflammation following hind-limb ischemia-reperfusion affects brain in neonatal mice, Dev. Neurosci. 30 (6) (2008) 367e373. [22] J. Lai, Y. Liu, C. Liu, et al., Indirubin inhibits LPS-induced inflammation via TLR4 abrogation mediated by the NF-kB and MAPK signaling pathways, Inflammation 40 (1) (2017) 1e12. [23] Y. Min, M.J. Kim, S. Lee, et al., Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation, Autophagy 14 (8) (2018) 1347e1358. [24] T. Shichita, E. Hasegawa, A. Kimura, et al., Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain, Nat. Med. 18 (6) (2012) 911. [25] W. Mei, Z. Peng, M. Lu, et al., P eroxiredoxin 1 inhibits the oxidative stress induced apoptosis in renal tubulointerstitial fibrosis, Nephrology 20 (11) (2015) 832e842. [26] J.W. Chang, S.H. Lee, J.Y. Jeong, et al., Peroxiredoxin-I is an autoimmunogenic tumor antigen in non-small cell lung cancer, FEBS Lett. 579 (13) (2005) 2873e2877. [27] J.W. Chang, S.H. Lee, Y. Lu, et al., Transforming growth factor-b1 induces the non-classical secretion of peroxiredoxin-I in A549 cells, Biochem. Biophys. Res. Commun. 345 (1) (2006) 118e123. [28] R. Jin, G. Yang, G. Li, Inflammatory mechanisms in ischemic stroke: role of inflammatory cells, J. Leukoc. Biol. 87 (5) (2010) 779e789. [29] Z. Tang, Y. Gan, Q. Liu, et al., CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke, J. Neuroinflammation 11 (1) (2014) 26. [30] Y. Sun, H. Gui, Q. Li, et al., MicroRNA-124 protects neurons against apoptosis
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461 in cerebral ischemic stroke, CNS Neurosci. Ther. 19 (10) (2013) 813e819. [31] G. Liu, T. Wang, T. Wang, et al., Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats, Biomedical reports 1 (6) (2013) 861e867. [32] P. Aguilar-Melero, M.J. Prieto-Alamo, J. Jurado, et al., Proteomics in HepG2 hepatocarcinoma cells with stably silenced expression of PRDX1, Journal of proteomics 79 (2013) 161e171. [33] L. Wang, Z. Li, X. Zhang, et al., Protective effect of shikonin in experimental ischemic stroke: attenuated TLR4, p-p38MAPK, NF-kB, TNF-a and MMP-9
461
expression, up-regulated claudin-5 expression, ameliorated BBB permeability, Neurochem. Res. 39 (1) (2014) 97e106. [34] J. Zhang, B. Fu, X. Zhang, et al., Neuroprotective effect of bicyclol in rat ischemic stroke: down-regulates TLR4, TLR9, TRAF6, NF-kB, MMP-9 and upregulates claudin-5 expression, Brain Res. 1528 (2013) 80e88. [35] J.R. Riddell, W. Bshara, M.T. Moser, et al., Peroxiredoxin 1 controls prostate cancer growth through Toll-like receptor 4edependent regulation of tumor vasculature, Cancer Res. 71 (5) (2011) 1637e1646.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
PRDX1 enhances cerebral ischemia-reperfusion injury through activation of TLR4-regulated inflammation and apoptosis Qiang Liu a, Yuan Zhang b, * a b
Department of Neurology, Yan'an University Affiliated Hospital, Yan'an, Shannxi, 716000, China Department of EMG Evoked Potential Chamber, Heze Municipal Hospital, Shandong Province, Heze City, Shandong Province, 274000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 August 2019 Accepted 13 August 2019 Available online 13 September 2019
Stroke is still a leading cause of death across the world. Despite various signals or molecules that contribute to the pathophysiological process have been investigated, the exact molecular mechanisms revealing stroke damage still remain to be explored. Peroxiredoxin 1 (PRDX1) has been identified as a stress-induced macrophage redox protein with multiple functions. Although PRDX1 is a critical factor related to the regulation of immunity, inflammation, apoptosis and oxidative stress, its effects on cerebral ischemia-reperfusion (I-R) injury were presently unclear. In the study, by using a mouse model of I-R injury, we found that PRDX1 expression was up-regulated during I-R injury in a time-dependent manner. Additionally, PRDX1-knockout mice showed reduced infarction area and alleviated neuropathological scores with decreased brain water contents. Furthermore, cell death and inflammatory response in mice with cerebral I-R injury were markedly attenuated by PRDX1 knockout, which were associated with the blockage of Caspase-3 and nuclear factor-kB (NF-kB) signaling pathways. Mechanistically, PRDX1regulated cerebral I-R injury was through the promotion of toll-like receptor-4 (TLR4), as proved by the evidence that TLR4 suppression abrogated the exacerbated effect of TLR4 on inflammatory response and apoptosis in oxygen and glucose deprivation (OGD)-treated primary microglial cells. These data demonstrated that PRDX1 contributed to cerebral stroke by interacting with TLR4, providing an effective therapeutic approach for cerebral I-R injury. © 2019 Published by Elsevier Inc.
Keywords: PRDX1 Cerebral ischemia-reperfusion injury Apoptosis Inflammation TLR4
1. Introduction Stroke is a major cause leading to death worldwide although advancements have been made in therapeutic approaches, and ischemic stroke accounts for approximate 85% of total stroke [1]. Excessive experimental and clinical studies have indicated that multiple pathophysiological processes are involved in cerebral I-R injury, such as immune response, inflammation, apoptotic cell death, endoplasmic reticulum stress and oxidative stress [2e4]. However, therapeutic strategies based on these molecular mechanisms showed unsatisfied outcomes [5]. Therefore, further studies are still warranted to investigate underlying mechanisms for the treatment of cerebral I-R damage. Peroxiredoxins (PRDXs), as important antioxidant proteins, constitute the potent defense system to sustain redox balance through converting hydrogen peroxide to water [6]. PRDX1 belongs
* Corresponding author. E-mail address: [email protected] (Y. Zhang). https://doi.org/10.1016/j.bbrc.2019.08.077 0006-291X/© 2019 Published by Elsevier Inc.
to PRDXs family, and has been reported as a 23-kDa stress-induced macrophage redox protein with a variety of functions [7,8]. Extracellular PRDX1 plays an essential role in regulating inflammatory response [9]. PRDX1 can function as a molecular chaperone with the ability to control the actions of multiple molecules or as a modulator for transcription [10]. PRDX1 expression during the early phase of extracorporeal membrane oxygenation (ECMO) support in cardiogenic shock patients is involved in the progression of systemic inflammatory response syndrome, accompanied with poor clinical outcomes [11]. Recently, PRDX1 was suggested to be a novel damage-associated molecular pattern, contributing to the development of acute liver injury [12]. Moreover, PRDX1 was involved in the progression of myocardial ischemia reperfusion injury by regulating cell apoptosis [13]. Although these initial clues have been elucidated, the exact effects of PRDX1 on cerebral I-R injury remain unclear. In this study, we found that the expression levels of PRDX1 were significantly up-regulated in cerebral I-R injury mouse model, and in microglial cells treated with OGD. PRDX1 knockout mice displayed alleviated infarction area and neurological scores with
454
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
2. Materials and methods
incubated with the primary antibodies overnight at 4 C, including PRDX1 (ab41906, dilution at 1:150, Abcam, USA), Iba1 (ab178847, dilution at 1:150, Abcam) and TLR4 (ab22048, dilution at 1:100, Abcam). After washing, sections were incubated with appropriate secondary antibodies (Abcam), followed by nuclei staining with DAPI (Sigma Aldrich). TUNEL (Terminal Dexynucleotidyl Transferase(Tdt)-Mediated Dutp Nick End Labeling) apoptosis assay kit was used to determine cell death following the manufacturer's protocol (Beyotime).
2.1. Animals and cerebral I-R injury
2.5. In vitro model for cerebral I-R
All animal procedures were performed according to the institutional guidelines for the Care and Use of Laboratory Animals of Yan'an University Affiliated Hospital (Shan'xi, China). All animal experiments were conducted on 10-12 week-old male mice. PRDX1 knock-out (KO) mice were purchased from Cyagen Biotech (Suzhou, China). A cerebral I-R model was established according to transient blockage of the proximal middle cerebral artery as previously indicated [14]. Briefly, mice were anesthetized using 3% isoflurane. After surgical exposure of the left carotid artery, a 6-0 siliconcoated monofilament was then inserted into the internal carotid artery through the external carotid artery, temporarily blocking blood flow to the middle cerebral artery (MCA). Following blockage for 45 min, the monofilament was carefully retracted from the internal carotid artery. Mice were kept immobilized for 10 min. Throughout the surgery, left cerebral blood flow was monitored using a Doppler flowmeter (Perimed, Sweden). For sham mice, the monofilament was withdrawn immediately after the initial onset of reduced cerebral blood flow. Rectal temperature was kept at 37 ± 0.5 C throughout the operation. All animals were allowed to recover in a 37 C incubator for different time periods with free access to food and water.
Primary microglia were isolated from WT and PRDX1-KO mice on postnatal days 0e2 as previously described by the published protocols [17]. Prior to treatment, microglia from mice were plated in chamber slides (100,000 cells/well) and allowed to adhere for 1 h in serum-free and glutamate-free DMEM/F12 nutrient mixture (Gibco, USA). To mimic I-R conditions in vitro, the cells were exposed to transient OGD for 60 min and then returned to normal culture conditions for different periods [18]. Short interfering RNA (siRNA) and plasmids to over-express PRDX1 or TLR4 were transfected into microglial cells using the Lipofectamine 2000 reagent (Invitrogen, USA) following the manufacturer's instructions. siRNAS against TLR4 and PRDX1, and PRDX1 or TLR4 specific plasmid were synthesized by RiboBio (Guangzhou, China).
decreased brain water contents. Additionally, cerebral I-R injuryinduced apoptosis and inflammatory response were also attenuated in mice with PRDX1 deletion. Furthermore, we elucidated that PRDX1 could interact with TLR4 to regulate NF-kB and cell death, resulting in cerebral I-R damage. Thus, targeting PRDX1/TLR4 represented a potential and effective therapeutic approach against ischemic stroke.
2.2. Quantitative real-time PCR Total RNA was extracted from the ipsilateral striatum of mice using Tripure Isolation Reagent (Roche, USA) according to the manufacturer's instructions for RT-qPCR analysis following standard protocols as previously described [15]. Primer pairs for PCR were presented in Supplement Table 1. 2.3. Western blot analysis Proteins extracted from the ipsilateral striatum of mice using RIPA lysis buffer (Beyotime, Shanghai, China) with protease inhibitor cocktail tablets. The protein expression levels were then measured using primary antibodies (Supplement Table 2) as previously described [16]. Protein expression levels were normalized to GAPDH. 2.4. Immunofluorescence and TUNEL staining For histopathological analysis, animals were euthanized and transcardially perfused with PBS (pH 7.4), followed by 4% paraformaldehyde. Then, brains were slowly removed and immersed in 4% paraformaldehyde for 2 h. Next, the brains were dehydrated using 20% sucrose solution in PBS overnight, followed by 30% sucrose for another 6 h. Subsequently, brains were embedded in Optimal Cutting Temperature Compound. 8-mm-thickness brain sections were made with a cryostat microtome. For antigen retrieval, brain slides were incubated in EDTA at 86 C for 5 min. For primary microglial cells, coverslips were fixed with 4% paraformaldehyde and then incubated in 10% goat serum (Sigma Aldrich, USA). For immunofluorescence labeling, all slides were
2.6. Fluoro-Jade B staining Fluoro-Jade B (Millipore) was used for cell death analysis in brain sections according to the manufacturer's protocols. 2.7. Analysis of infarct size The brains were removed for infract volume calculation through the 2,3,5-triphenyltetrazolium chloride (TTC, Sigma Aldrich) staining. Brain sections were stained in 2% TTC for 30 min at 37 C, and fixed in 10% formaldehyde neutral buffer overnight. The infract tissues were unstained, and the red regions was served as the normal area. 2.8. Neurological impairment score After reperfusion for 24 h, neurological impairment resulted from the tMCAO procedure was calculated according to the ninepoint scale [19]. The investigators were blinded to all measurements of the neurological deficits. 2.9. Brain water content analysis Brains were removed to determine wet weight immediately. Then, the brain samples were dried in an oven at 110 C for 48 h to measure the dry weight. Brain water content was determined as (wet weight-dry weight)/wet weight100%. 2.10. Evans blue extravasation To determine the blood-brain barrier (BBB) permeability at 24 h post-surgery, mice were treated with 100 ml of 4% Evans blue dye (Sigma Aldrich) via retro-orbital injection. After 2 h, we perfused the mouse transcardially with saline, removed the brain. 2.11. Flow cytometry analysis Apoptosis was calculated by fluorescein isothiocyanate (FITC)conjugated Annexin V and propidium iodide (PI) staining
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
455
Fig. 1. PRDX1 expression levels were induced after experiencing cerebral I-R injury in vivo and in vitro. (A) RT-qPCR and (B) western blotting analysis demonstrating the expression levels of PRDX1 in mice after cerebral I-R injury at the indicated time. (C) Immunofluorescent staining of PRDX1 in striatum of mice after cerebral I-R injury. (D) RT-qPCR, (E) western blotting and (F) immunofluorescent analysis indicating the expression levels of PRDX1 in primary isolated microglial cells treated with OGD at the indicated time points. (G) Representative western blot of PRDX1 levels in brain tissues of WT mice and PRDX1-KO mice. (H) Representative images of TTC staining of brain sections from both WT and PRDX1-KO groups after reperfusion for 24 h. Quantitative analysis of (I) infarct volume and (J) neurological scores of the mice from the indicated groups. (K) Representative images of Evans blue extravasation in peri-infarction area at 24 h after cerebral I-R injury. (L) Quantification of Evans blue leakage in mice after cerebral I-R injury for 24 h. (M) Brain water content was examined at 24 h after cerebral I-R injury. Data were expressed as mean ± SEM (n ¼ 8). *P < 0.05, **P < 0.01 and ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
456
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
Fig. 2. PRDX1 knockout attenuated ischemia-reperfusion injury. (A) Fluoro-Jade B and TUNEL staining of brain sections in mice. Quantification of Fluoro Jade B- and TUNELpositive cells were exhibited. (B) Western blot analysis of Bcl-2, Bax, Bid and cleaved Caspase-3 expression levels in the ipsilateral striatum of mice. (C) TUNEL staining of primary microglial cells treated with OGD for 24 h. TUNEL-positive cells were quantified. (D,E) Flow cytometry analysis of primary microglial cells exposed to 24 h of OGD. Apoptosis rate was analyzed. (F) Western blot analysis for Bcl-2 and Caspase-3 cleavage in primary microglial cells treated with OGD for 24 h. Data were expressed as mean ± SEM (n ¼ 8). * P < 0.05, **P < 0.01 and ***P < 0.001.
(Beyotime) according to the manufacturer's instructions. 2.12. Immunoprecipitation (IP) and GST pull-down Human PRDX1 cDNA was cloned into psi-Flag-C1 and pcDNA5HA-C1 to express Flag-PRDX1, HA-PRDX1 and GST-HA-PRDX1 fusion protein, respectively. TLR4-expressing vectors were created
by the same method. IP analysis of PRDX1 and TLR4 were conducted to calculate the interaction of each protein under normal and OGD conditions as previously described [16,18]. GST Pull-down analysis was conducted to determine the direct binding between purified PRDX1 and TLR4 proteins also as previously indicated [16,18].
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
457
Fig. 3. PRDX1 knockout alleviated inflammatory response after ischemia-reperfusion injury. RT-qPCR results for (A) Iba1, (B) TNF-a, COX2, IL-1b, IL-6 and IL-10 in the ipsilateral striatum of mice. (C) Western blot analysis for p-IkBa and p-NF-kB in the ipsilateral striatum of mice. (D) Immunofluorescent staining of Iba-1 and TLR-4 in brain sections of mice from the indicated groups. (E) Western blot analysis for TLR4 and MyD88 in the ipsilateral striatum of mice. (F) Western blot analysis for TLR4, MyD88, p-IkBa and p-NF-kB in primary microglial cells treated with OGD for 24 h. Data were expressed as mean ± SEM (n ¼ 8). *P < 0.05, **P < 0.01 and ***P < 0.001.
2.13. Statistical analysis
3. Results
Data were expressed as mean ± SEM using Graph Pad Prism 6.0 software. One-way analysis of variance (ANOVA) was performed followed by Bonferroni's post-hoc analysis between multiple groups. Comparisons between two groups were performed by Student's t-test. Value of p < 0.05 was considered statistically significant.
3.1. PRDX1 expression levels were enhanced after experiencing cerebral I-R injury in vivo and in vitro In this part, we first found that PRDX1 expression from mRNA and protein levels detected by RT-qPCR and western blotting were markedly increased in a time-dependent manner after cerebral I-R
458
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
Fig. 4. PRDX1-induced ischemia-reperfusion injury via TLR4 activation. (A) IP with an HA antibody or control was performed for western blot using a Flag antibody (up panel) or IP with a Flag antibody for western blot with an HA antibody (down panel). (B) GST pull-down analysis in which either GST-tagged PRDX1 (HA-GST-PRDX1) or control HA-GST was used to pull down Flag-TLR4 (left panel) or GST-tagged TLR4 (HA-GST-TLR4) or HA-GST was used to pull down Flag-PRDX1 (right panel). (C) Primary microglial cells were
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
injury (Fig. 1A and B). Immunofluorescent staining suggested that cerebral I-R led to a significant increase in PRDX1-positive microglia in striatum of mice (Fig. 1C). In vitro, the isolated primary microglia treated with OGD showed higher expression levels of PRDX1 also in a time-dependent fashion (Fig. 1D and E). Similar expression change of PRDX1 was observed in OGD-incubated primary microglial cells using immunofluorescent staining (Fig. 1F). Then, to further investigate if PRDX1 played a critical role in modulating cerebral I-R injury, PRDX1-KO mice were used, and identified by western blot analysis (Fig. 1G). After MCAO, PRDX1-KO mice showed remarkable reductions in infarction area and neurological scores (Fig. 1HeJ). BBB permeability at 24 h after MCAO was then assessed using Evans blue extravasation. As shown in Fig. 1K and L, Evans blue leakage was markedly decreased in MCAO mice with PRDX1 deletion. Finally, MCAO-enhanced brain water contents were highly decreased by PRDX1 knockout (Fig. 1M). Together, the findings above strongly demonstrated a critical role for PRDX1 in regulating stroke damage. 3.2. PRDX1 knockout attenuated ischemia-reperfusion injury Fluorescent staining suggested that PRDX1-KO mice showed a sharp decrease in Fluoro Jade B- and TUNEL-positive cells after MCAO operation in comparison with the WT mice (Fig. 2A). In PRDX1-KO mice after MCAO for 24 h, up-regulation in the levels of the pro-apoptotic proteins, including Bax, Bid and cleaved Caspase3, were lower than that in the WT mice. In contrast, expression of the anti-apoptotic factor Bcl2 was higher than that in the WT group of mice following I-R injury (Fig. 2B). In vitro, primary microglial cells subjected to OGD also showed significant downregulation of TUNEL-positive cells with PRDX1 deficiency (Fig. 2C). Flow cytometry analysis also demonstrated that OGDinduced apoptosis in primary microglia was greatly alleviated by PRDX1 ablation (Fig. 2D and E). Results by western blotting suggested that anti-apoptotic signal Bcl-2 expression levels were markedly rescued by PRDX1 knockout in OGD-stimulated microglial cells. However, opposite results were detected in the expression change of cleaved Caspase-3 (Fig. 2F). 3.3. PRDX1 knockout alleviated inflammatory response after ischemia-reperfusion injury Microglial activation and inflammation play critical roles in the development of cerebral I-R injury [20,21]. Here, the mRNA levels of Iba1, a critical microglial marker, were significantly induced by MCAO, while being reduced in the PRDX1-KOgroup (Fig. 3A). Then, we found that the mRNA levels for pro-inflammatory regulators, such as TNF-a, COX2, IL-1b and IL-6, were markedly reduced in the PRDX1-KO group after MCAO operation, while anti-inflammatory modulator IL-10 mRNA levels were restored (Fig. 3B). Moreover, western blotting demonstrated that the phosphorylation of IkBa and NF-kB (p65) was clearly reduced in PRDX1-KO mice (Fig. 3C). TLR4 is of great importance in regulating inflammatory response via NF-kB signaling [22]. In addition, PRDX1 was reported to meditate TLR4 signaling under various conditions [23]. Then, immunofluorescent staining demonstrated that TLR4 expression levels stimulated by MCAO were markedly alleviated by PRDX1
459
deletion (Fig. 3D). Consistently, PRDX1-KO mice exhibited significantly reduced the expression levels of TLR4 and its downstreaming signal MyD88 following MCAO (Fig. 3E). As expected, TLR4, MyD88, phosphorylation levels of IkBa and NF-kB stimulated by OGD in primary microglial cells were greatly attenuated by the PRDX1 deletion (Fig. 3F). Altogether, these results indicated that, following MCAO, PRDX1 contributed to the pathological inflammation that resulted in cerebral I-R injury. 3.4. PRDX1-induced ischemia-reperfusion injury directly dependent on TLR4 activation We further investigated the interaction of PRDX1 and TLR4 to explore the molecular mechanism of TLR4 suppression by PRDX1 deletion in cerebral I/R injury. HA-tagged PRDX1 and Flag-tagged TLR4 were overexpressed in 293T cells. IP analysis indicated that PRDX1 co-immunoprecipitated with TLR4 and vice versa regardless of the OGD insult (Fig. 4A). A GST-tagged PRDX1 effectively pulled down TLR4, and GST-tagged TLR4 could pull down PRDX1 (Fig. 4B). Thus, we supposed that PRDX1 could directly interact with TLR4. To further reveal if PRDX1-induced cerebral injury was TLR4dependent, primary microglial cells with PRDX1 and TLR4 knockdown and/or over-expression were generated (Fig. 4C). When microglial cells were exposed to OGD, however, co-transfection with siTLR4 counteracted oePRDX1-induced expression of proinflammatory cytokines TNF-a, COX2, IL-1b and IL-6 (Fig. 4D) and apoptosis (Fig. 4E). Conversely, siPRDX1-reduced inflammation and apoptosis were clearly abolished by TLR4 over-expression in OGDexposed microglial cells (Fig. 4F and G). PRDX1-induced cerebral I-R injury was largely TLR4-dependent. 4. Discussion Ischemic stroke is considered as a leading global cause for mortality and disability [1e3]. Despite multiple pathological processes have been targeted to improve ischemic brain injury, effective translation of these findings into clinical treatment still require further studies to reveal the underlying molecular mechanisms. In the present study, PRDX1 might be an up-streaming modulator to regulate inflammation and apoptosis after MCAO. Importantly, PRDX1/TLR4 axis showed a potential role in controlling cerebral I-R damage (Fig. 4H). The most significant finding of our present study is that PRDX1 could interact with TKR4 to regulate NF-kB and apoptosis, leading to cerebral I-R injury. PRDX1 is a small protein (23 kDa) in the Peroxiredoxins family, which is composed of ubiquitously expressed enzymes that down-regulate peroxide levels [5,6]. PRDX family proteins are key initiators of post-ischemic inflammation in the brain [24]. PRDX1 is well-known for its protective effects on different diseases including aging, neurodegenerative diseases and cancers due to its anti-oxidative capability [25]. However, a significant increase of PRDX1 in serum of patients with non-small cell lung cancer was discovered [26]. Moreover, intracellular PRDX1 is released to the extracellular space when respond to stimuli such as TGF-b1, LPS and TNF-a [27]. Recently, a significant promotion of circulating PRDX1 in mice with acute liver injury was discovered, which is associated with the release of pro-
transfected with PRDX1 siRNA, PRDX1 plasmid, TLR4 siRNA or TLR4 plasmid to inhibit or promote PRDX1 and TLR4 expressions, respectively, for 24 h. Then, all cells were harvested for transfection efficacy calculation using western blot analysis. (D,E) Primary microglial cells were transfected with siTLR4, oePRDX1 or the two in combination for 24 h. Then, these cells were subjected to OGD treatment for another 24 h. Next, (D) RT-qPCR analysis was used to determine TNF-a, COX2, IL-1b, IL-6 and IL-10 mRNA levels in cells. (E) Flow cytometry analysis was used to measure apoptosis. (F,G) Primary microglia were transfected with oeTLR4, siPRDX1 or the two in combination for 24 h. Next, the cells were treated with OGD for another 24 h. Subsequently, (F) TNF-a, COX2, IL-1b, IL-6 and IL-10 mRNA levels in cells were assessed using RT-qPCR analysis. (G) Flow cytometry analysis was conducted to evaluate apoptosis. (H) Diagram depicting the interaction between PRDX1 and TLR4 that resulted in cerebral I-R injury. Data were expressed as mean ± SEM (n ¼ 4). * P < 0.05, **P < 0.01 and ***P < 0.001.
460
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461
inflammatory cytokines such as IL-1b, IL-6 and TNF-a through activating NF-kB signaling; however, mice with PRDX1 deficiency were protected from acute liver injury and its associated inflammatory response [12]. Inflammatory response plays an important role after cerebral ischemia. Brain damage results in activation of resident microglia after ischemia, which is followed by infiltration of immunocytes, such as neutrophils, T cells, and macrophages [28]. A variety of pro-inflammatory regulators are induced including TNF-a, COX2, IL-1b and IL-6, accelerating brain damage. Suppressing microglia activation could represent an effective therapeutic target to alleviate acute cerebral ischemia [29]. Here, in the present study, we first found that PRDX1 expression in microglial cells was significantly induced by cerebral I-R injury both in vivo and in vitro. Then, PRDX1-KO mice exhibited alleviated cerebral I-R injury induced by MCAO, which was largely associated with the suppression of pro-inflammatory cytokines (TNF-a, COX2, IL-1b and IL-6) and improvement of anti-inflammatory factor (IL10). Similar inhibitory effects of PRDX1 deletion on inflammatory response were confirmed in OGD-treated primary microglial cells, and these effects were attributed to the inactivation of NF-kB signaling. Apoptosis plays a critical role in the pathophysiology of cerebral hypoperfusion. Bcl-2, Bax and Bid are representatively proteins; Caspase-3 is another crucial factor that contributes to the progression of apoptosis [30]. Bcl-2 is important for anti-apoptosis, while Bax and Bis are key pro-apoptotic signals. The ratio of Bcl2/Bax is proposed to determine the apoptotic fate of the cell [31]. In this work, we confirmed that MCAO led to apoptosis in the injured brain samples, as evidenced by the reduced expression levels of Bcl-2, and enhanced levels of Bax, Bid and subsequent Caspase-3 cleavage. Notably, this pro-apoptotic process was greatly blunted by PRDX1 knockout, attenuating cerebral I-R injury. PRDX1 may contribute to apoptosis inhibition in hepatic cancer cells under normal non-stressed conditions [32]. Thus, PRDX1-regulated cerebral I-R injury was also associated with its regulation to apoptotic condition. TLR4 is a key PRR, which binds and contacts down-streaming NF-kB through receptor dimerization. Activated components of the NF-kB signaling pathway meditate inflammation by promoting the expression of TNF-a, COX2, IL-1b and IL-6, as well as other proinflammatory cytokines or chemokine [33]. Suppressing the activation of the TLR4 signaling is a potential anti-inflammatory treatment and may prevent ischemic stroke [34]. Unexpectedly, extracellular PRDX1 has recently been reported as a novel damageassociated molecular pattern (DAMP) attributed to its proinflammatory property through binding to TLR4 [35]. In this study, we not only elucidated that PRDX1 regulated TLR4 expression and the subsequent inflammatory cytokine production during the ischemic I-R injury, but also demonstrated that the promoting effects of PRDX1 on the activation NF-kB signaling and apoptosis depended on the interaction and activation of TLR4. These data provided a therapeutic insight into the PRDX1/TLR4 regulatory axis during the progression of cerebral I-R injury. In summary, findings in this work illustrated a novel function of PRDX1 in regulating ischemic stroke through interacting with TLR4, which subsequently promoted inflammatory response and apoptosis through NF-kB and Caspase-3, respectively (Fig. 4H). Herein, targeting PRDX1 could be insightful for developing new treatments against ischemic stroke for patients.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.077.
References [1] V.L. Feigin, B. Norrving, G.A. Mensah, Global burden of stroke, Circ. Res. 120 (3) (2017) 439e448. [2] T.C. Burns, G.K. Steinberg, Stem cells and stroke: opportunities, challenges and strategies, Expert Opin. Biol. Ther. 11 (4) (2011) 447e461. [3] A.K. Saenger, R.H. Christenson, Stroke biomarkers: progress and challenges for diagnosis, prognosis, differentiation, and treatment, Clin. Chem. 56 (1) (2010) 21e33. [4] T.C. Burns, G.K. Steinberg, Stem cells and stroke: opportunities, challenges and strategies, Expert Opin. Biol. Ther. 11 (4) (2011) 447e461. [5] M.A. Yenari, T.M. Kauppinen, R.A. Swanson, Microglial activation in stroke: therapeutic targets, Neurotherapeutics 7 (4) (2010) 378e391. [6] Y. Zhou, S. Duan, Y. Zhou, et al., Sulfiredoxin-1 attenuates oxidative stress via Nrf2/ARE pathway and 2-Cys Prdxs after oxygen-glucose deprivation in astrocytes, J. Mol. Neurosci. 55 (4) (2015) 941e950. [7] B. Turner-Ivey, Y. Manevich, J. Schulte, et al., Role for Prdx1 as a specific sensor in redox-regulated senescence in breast cancer, Oncogene 32 (45) (2013) 5302. ant, C. Che ry, A. Oussalah, et al., A PRDX1 mutant allele causes a [8] J.L. Gue MMACHC secondary epimutation in cblC patients, Nat. Commun. 9 (1) (2018) 67. [9] D. Liu, P. Mao, Y. Huang, et al., Proteomic analysis of lung tissue in a rat acute lung injury model: identification of PRDX1 as a promoter of inflammation, Mediat. Inflamm. 2014 (4) (2014), 469358. [10] J. Cao, J. Schulte, A. Knight, et al., Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity, EMBO J. 28 (10) (2009) 1505e1517. [11] C.H. Liu, S.W. Kuo, L.M. Hsu, et al., Peroxiredoxin 1 induces inflammatory cytokine response and predicts outcome of cardiogenic shock patients necessitating extracorporeal membrane oxygenation: an observational cohort study and translational approach, J. Transl. Med. 14 (1) (2016) 114. [12] Y. He, S. Li, D. Tang, et al., Circulating Peroxiredoxin-1 is a novel damageassociated molecular pattern and aggravates acute liver injury via promoting inflammation, Free Radic. Biol. Med. 137 (2019) 24e36. [13] W. Guo, X. Liu, J. Li, et al., Prdx1 alleviates cardiomyocyte apoptosis through ROS-activated MAPK pathway during myocardial ischemia/reperfusion injury, Int. J. Biol. Macromol. 112 (2018) 608e615. [14] T. Li, J.J. Qin, X. Yang, et al., The ubiquitin E3 ligase TRAF6 exacerbates ischemic stroke by ubiquitinating and activating Rac1, J. Neurosci. 37 (50) (2017) 12123e12140. [15] L. Wang, L. Wang, Z. Dai, et al., Lack of mitochondrial ferritin aggravated neurological deficits via enhancing oxidative stress in a traumatic brain injury murine model, Biosci. Rep. 37 (6) (2017). BSR20170942. [16] S. Wang, Z.Z. Yan, X. Yang, et al., Hepatocyte DUSP14 maintains metabolic homeostasis and suppresses inflammation in the liver, Hepatology 67 (4) (2018) 1320e1338. [17] J.M. Crain, M. Nikodemova, J.J. Watters, Microglia express distinct M1 and M2 phenotypic markers in the postnatal and adult central nervous system in male and female mice, J. Neurosci. Res. 91 (9) (2013) 1143e1151. [18] Y.Y. Lu, Z.Z. Li, D.S. Jiang, et al., TRAF1 is a critical regulator of cerebral ischaemiaereperfusion injury and neuronal death, Nat. Commun. 4 (2013) 2852. [19] C.F. Xia, R.S. Smith Jr., B. Shen, et al., Postischemic brain injury is exacerbated in mice lacking the kinin B2 receptor, Hypertension 47 (4) (2006) 752e761. [20] M. Lv, Y. Liu, J. Zhang, et al., Roles of inflammation response in microglia cell through Toll-like receptors 2/interleukin-23/interleukin-17 pathway in cerebral ischemia/reperfusion injury, Neuroscience 176 (2011) 162e172. [21] M.D. Bianco-Batlles, A. Sosunov, R.A. Polin, et al., Systemic inflammation following hind-limb ischemia-reperfusion affects brain in neonatal mice, Dev. Neurosci. 30 (6) (2008) 367e373. [22] J. Lai, Y. Liu, C. Liu, et al., Indirubin inhibits LPS-induced inflammation via TLR4 abrogation mediated by the NF-kB and MAPK signaling pathways, Inflammation 40 (1) (2017) 1e12. [23] Y. Min, M.J. Kim, S. Lee, et al., Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation, Autophagy 14 (8) (2018) 1347e1358. [24] T. Shichita, E. Hasegawa, A. Kimura, et al., Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain, Nat. Med. 18 (6) (2012) 911. [25] W. Mei, Z. Peng, M. Lu, et al., P eroxiredoxin 1 inhibits the oxidative stress induced apoptosis in renal tubulointerstitial fibrosis, Nephrology 20 (11) (2015) 832e842. [26] J.W. Chang, S.H. Lee, J.Y. Jeong, et al., Peroxiredoxin-I is an autoimmunogenic tumor antigen in non-small cell lung cancer, FEBS Lett. 579 (13) (2005) 2873e2877. [27] J.W. Chang, S.H. Lee, Y. Lu, et al., Transforming growth factor-b1 induces the non-classical secretion of peroxiredoxin-I in A549 cells, Biochem. Biophys. Res. Commun. 345 (1) (2006) 118e123. [28] R. Jin, G. Yang, G. Li, Inflammatory mechanisms in ischemic stroke: role of inflammatory cells, J. Leukoc. Biol. 87 (5) (2010) 779e789. [29] Z. Tang, Y. Gan, Q. Liu, et al., CX3CR1 deficiency suppresses activation and neurotoxicity of microglia/macrophage in experimental ischemic stroke, J. Neuroinflammation 11 (1) (2014) 26. [30] Y. Sun, H. Gui, Q. Li, et al., MicroRNA-124 protects neurons against apoptosis
Q. Liu, Y. Zhang / Biochemical and Biophysical Research Communications 519 (2019) 453e461 in cerebral ischemic stroke, CNS Neurosci. Ther. 19 (10) (2013) 813e819. [31] G. Liu, T. Wang, T. Wang, et al., Effects of apoptosis-related proteins caspase-3, Bax and Bcl-2 on cerebral ischemia rats, Biomedical reports 1 (6) (2013) 861e867. [32] P. Aguilar-Melero, M.J. Prieto-Alamo, J. Jurado, et al., Proteomics in HepG2 hepatocarcinoma cells with stably silenced expression of PRDX1, Journal of proteomics 79 (2013) 161e171. [33] L. Wang, Z. Li, X. Zhang, et al., Protective effect of shikonin in experimental ischemic stroke: attenuated TLR4, p-p38MAPK, NF-kB, TNF-a and MMP-9
461
expression, up-regulated claudin-5 expression, ameliorated BBB permeability, Neurochem. Res. 39 (1) (2014) 97e106. [34] J. Zhang, B. Fu, X. Zhang, et al., Neuroprotective effect of bicyclol in rat ischemic stroke: down-regulates TLR4, TLR9, TRAF6, NF-kB, MMP-9 and upregulates claudin-5 expression, Brain Res. 1528 (2013) 80e88. [35] J.R. Riddell, W. Bshara, M.T. Moser, et al., Peroxiredoxin 1 controls prostate cancer growth through Toll-like receptor 4edependent regulation of tumor vasculature, Cancer Res. 71 (5) (2011) 1637e1646.