- Email: [email protected]
Foxo1-mediated inflammatory response after cerebral hemorrhage in rats
Neuroscience Letters 629 (2016) 131–136
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Foxo1-mediated inflammatory response after cerebral hemorrhage in rats Li Zhenyu a , He Qi a , Zhai Xuan c , You Yan a , Li Lingyu b , Hou Yanghao a , He Faming d , Zhao Yong b , Zhao Jing a,∗ a
Department of Pathophysiology, Chongqing Medical University, Chongqing, People’s Republic of China Department of Pathology, Chongqing Medical University, Chongqing, People’s Republic of China c Department of Neurosurgery, The Children’s Hospital of Chongqing Medical University, Chongqing, People’s Republic of China d Department of Emergency, The First Affiliated Hospital of Chongqing Medical University. Chongqing, People’s Republic of China b
h i g h l i g h t s • • • • •
Foxo1 expression peaked at 12 h post-intracerebral hemorrhage (ICH) and in the ipsilateral corpus striatum. Intracerebroventricular injection of Foxo1 siRNA effectively inhibited Foxo1 mRNA and protein expression. Foxo1 siRNA significantly increased neurological function and decreased brain water content after ICH injury. Foxo1 siRNA obviously reduced inflammatory factors release. Inhibition of Foxo1 may inhibited the TLR4/NF-B pathway after ICH injury.
a r t i c l e
i n f o
Article history: Received 20 April 2016 Received in revised form 30 May 2016 Accepted 6 June 2016 Available online 7 June 2016 Keywords: Foxo1 Inflammatory injury TLR4 Immunoregulation Intracerebral hemorrhage MPO
a b s t r a c t The forkhead box O (Foxo) family of transcription factors plays a crucial role in cell apoptosis, immune regulation, and tissue development. Foxo1, as the foremost member of the Foxo family, regulates a wide range of molecular signals in many tissues, including tumor, liver, and brain. This study investigated Foxo1 expression at different time points and in different brain areas, and the role of Foxo1 in vivo in regulating inflammatory injury in a rat model of autologous blood-injected cerebral hemorrhage injury. We found that Foxo1 expression peaked at 12 h post-intracerebral hemorrhage (ICH) and in the ipsilateral corpus striatum. Foxo1 knockdown by Foxo1 siRNA decreased ICH injury, improved neurological function, and decreased the expression of inflammatory factors downstream of the Foxo1 pathway, including TLR4, NF-B, TNF-␣, IL-1, and IL-18. Foxo1 knockdown also decreased the expression and activity of myeloperoxidase, IL-1, and IL-18. In conclusion, our findings demonstrate that Foxo1 is a key regulator of inflammatory injury in rats after ICH. By identifying the molecular mechanisms of Foxo1/TLR4/NF-B signaling, we provide a novel rationale for therapeutic approaches to managing inflammatory injury after ICH. © 2016 Published by Elsevier Ireland Ltd.
1. Introduction
Abbreviations: Foxo1, the forkhead box O1; ICH, intracerebral hemorrhage; siRNA, small interfering RNA; TLR4, toll-like receptor 4; NF-B, nuclear factork-gene binding; TNF-␣, tumor necrosis factor; IL-1, interleukin-1; IL-18, interleukin-18; MPO, myeloperoxidase; PASMSs, pulmonary artery smooth muscle cells; RT-PCR, reverse transcription-polymerase chain reaction; ELISA, enzymelinked immunosorbent assay; WW, wet weight; DW, dry weight; Ipsi-CS, ipsilateral corpus striatum; Ipsi-CX, ipsilateral cortex; Cont-CS, contralateral corpus striatum; Cont-CX, contralateral cortex. ∗ Corresponding author at: Department of Pathophysiology, Institute of Neuroscience, Chongqing Medical University, Yixueyuan Road 1, Chongqing, 400016, People’s Republic of China. E-mail address: [email protected] (J. Zhao). http://dx.doi.org/10.1016/j.neulet.2016.06.013 0304-3940/© 2016 Published by Elsevier Ireland Ltd.
Intracerebral hemorrhage (ICH), an important clinical classification of stroke, induces neuronal apoptosis, formation of perihematomal edema, inflammatory lesions, neurological impairment, and energy metabolism disorder [1]. ICH remains a tremendous challenge for which there is no effective therapy at present [2]. Cerebral edema starts with an inflammatory reaction following introduction of a hematoma. In particular, blood-derived leukocytes and neutrophils infiltrate the brain parenchyma, disrupting the blood–brain barrier and causing edema formation as well as deterioration of neurobehavioral function [3]. These
132
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
responses are consequences of increased levels of myeloperoxidase (MPO), IL-1, and IL-18 [4–6]. Therefore, it is important to find a key upstream transcription factor in the inflammatory reaction. The forkhead box O (Foxo) family of transcription factors plays a crucial role in cell apoptosis, inflammation, and tissue development. Foxo1, a member of the Foxo family, regulates a wide range of molecular signals in many tissue types, such as tumor, liver, and brain [7,8]. Previous studies have suggested that under inflammatory conditions, Foxo1 expression is upregulated, which could induce the production of cultured macrophage inflammatory factors [9]. Immunoregulation of Foxo1 was demonstrated by enhancing TLR4-mediated inflammation in lung and liver tissue, among others, but its immunoregulation function in brain tissue after ICH in rats was uncertain [10,11]. In this study, we investigated the role of Foxo1 in ICH using an experimental ICH model with Foxo1 knockdown. We evaluated whether downregulated Foxo1 could reduce brain damage. Furthermore, we hypothesized that Foxo1 could regulate inflammatory injury via the TLR4/NF-B signaling pathway and modulate inflammatory factor activity. 2. Material and methods 2.1. Animals The animal study was approved by the Institutional Animal Care and Use Committee at Chongqing Medical University. Eightweek-old male Sprague–Dawley rats (weight 280–320 g, sourced from the Chongqing Medical Animal Experimentation Center) were housed in a temperature- and light-controlled environment under pathogen-free conditions, and provided unlimited access to food and water, 12/12 light/dark with humidity 60 ± 5% and 22 ± 3 ◦ C. All rats were randomly allocated to the following group: sham surgery group, ICH group, Foxo1 siRNA group and control siRNA of Foxo1 group in blind manner.
siRNAs of Foxo1-rat-1612 were 5 -GCCAAGACCCACAUAAUCATT-3 (sense) and 5 -UGAUUAUGUGGGUCUUGGCTT-3 (antisense). All siRNA was synthesized, purified, desalted, and shipped in the 2 -deprotected, duplexed form. 2.4. Injection of Foxo1-rat-siRNA Each tube of Foxo1-rat-siRNA was dissolved in 20 L DEPC, oscillated, centrifuged, and oscillated again, then infused into the right paracele at anterior–posterior 1 mm, mediolateral 2 mm, and dorsoventral 3.5 mm (8 L per rat), and retained for 15 min. The ICH model was established 24 h later. The stable negative control and scramble siRNA of Foxo1-rat-1612 were administered using the same procedure. 2.5. Brain water content assay ICH rats were decapitated under deep anesthesia, and the brains were immediately removed. Each brain was divided into two halves: hemorrhagic (ipsilateral) and contralateral. Each part was weighed on an electronic analytical balance to give the wet weight (WW) and dried at 100 ◦ C for 24 h in the oven to determine the dry weight (DW). Brain water content was then calculated as [(WW − DW)/WW] × 100%. 2.6. Garcia neurological score assay [12] ICH rats were given a score between 0 and 18 on the Garcia test, which consists of six sections: spontaneous activity, axial sensation, vibrissae proprioception, limb outstretching, lateral turning, and forelimb walking, with a possible score of 0–3 for each section (0 = worst, 3 = best). 2.7. Western blot
ICH was induced with a single infusion model of autologous blood (50 L). The rats were randomly assigned to an experimental group, anesthetized with 10% chloral hydrate (1 mL/100 g) by intraperitoneal injection, and stabilized prone in a stereotactic head frame. The autologous blood was collected from the arteria femoralis on the operation side and transferred into an anticoagulation syringe with a microinjection pump. The blood was infused into the right corpus striatum anterior-posterior 0.2 mm, mediolateral 3.0 mm, and dorsoventral 5.8 mm. An injection of 10 L blood was delivered 5.0 mm dorsoventral and retained for 2 min; the final 40 L blood was given as a total injection at dorsoventral 5.8 mm and retained for 15 min. Sham-operated rats were only given a needle insertion.
Amounts of 30 g of protein from each brain sample were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Monoclonal rabbit antirat Foxo1 (C29H4, Cell Signaling Technology, Danvers, USA, 1:1000, AB 2106495, Rabbit Anti-Foxo1), TLR4 (BS3489, Bioworld Technology, Minnesota, USA, 1:500, AB 1662746, Rabbit AntiTLR4), NF-B (sc-372, Santa Cruz Biotechnology, Texas, USA, 1:1000, AB 632037, Rabbit Anti-NF-B), TNF-␣ (BS1857, Bioworld Technology, Minnesota, USA, 1:1000, AB1662107, Rabbit AntiTNF-␣), IL-1 (BS3506, Bioworld Technology, Minnesota, USA, 1:500, AB 1661842, Rabbit Anti-IL-1), IL-18 (sc-7954, Santa Cruz Biotechnology, Texas, USA, 1:200, AB 1564060, Rabbit Anti-IL18), and -actin (13E5, Cell Signaling Technology, Danvers, USA, 1:1000, AB 2223169, Rabbit Anti--actin) were used. Relative protein quantities were determined by densitometry and expressed in absorbance units.
2.3. Preparation of siRNA
2.8. Quantitative RT-PCR
The siRNA against Foxo1 was designed using RNA oligochemical synthesis (Genepharma, Shanghai, China). Four Foxo1-rat-siRNA fragments were used: Foxo1-rat1285 (sense: 5 -GCAGACACCUUGCUAUUCATT-3 , antisense: 5 -UGAAUAGCAAGGUGUCUGCTT-3 ); Foxo1-rat-1434 (sense: 5 -GAGGAUUGAACCAGUAUAATT-3 , antisense: 5 -UUAUACUGGUUCAAUCCUCTT-3 ); Foxo1-rat-1612 (sense: 5 -CCAGGCACCUCAUAACAAATT-3 , antisense: 5 UUUGUUAUGAGGUGCCUGGTT-3 ); and stable negative contol 5 -UUCUCCGAACGUGUCACGUTT-3 , antisense: 5 (sense: ACGUGACACGUUCGGAGAATT-3 ). The most significant fragment screened by western blot was Foxo1-rat-1612, and the scramble
Total RNA was extracted from frozen brain using Reagent Kit(TaKaRa Biotechnology, Dalian, China). A total of 40 L RNA was reverse-transcribed into cDNA. Quantitative PCR was performed as described [13]. Primer sequences for the amplification of Foxo1 and -actin. Target gene expressions were calculated by their ratios to -actin.
2.2. ICH rat model
2.9. Enzyme-linked immunosorbent assay The presence of MPO was used as an index of neutrophil accumulation in the post-ICH brain tissue homogenate. The presence of IL-1 and IL-18 was used as an index of inflammatory injury in
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
133
Fig. 1. Foxo1 expression at different time points and brain areas after intracerebral hemorrhage (ICH). (A) Western blot assay for profiles of Foxo1 expression in sham, 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h ICH rats. (B) Quantification of Foxo1 expression in the ipsilateral corpus striatum in sham, 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h ICH rats. (12 h vs. other groups, **P < 0.01, n = 4 per group). (C) Western blot assay for profiles of Foxo1 expression in the ipsilateral corpus striatum and cortex, or contralateral corpus striatum and cortex in 12 h ICH. (D) Quantification of Foxo1 expression in the ipsilateral corpus striatum and cortex, or contralateral corpus striatum and cortex in 12 h ICH rats (ipsilateral corpus striatum vs. other groups, **P < 0.01, n = 4 per group). (E) Quantitative RT-PCR assay for mRNA level of Foxo1 in the ipsilateral corpus striatum and cortex, or contralateral corpus striatum and cortex, in 12 h ICH rats; data were normalized to -actin gene (ipsilateral corpus striatum vs. other groups, **P < 0.01, n = 4 per group; data represented as mean ± standard error).
the post-ICH brain tissue homogenate. MPO, IL-1, and IL-18 activity levels were assayed by ELISA kits(Boster Biological Technology, Wuhan, China) as previously described [14]. 2.10. Statistics Data were expressed as mean ± standard error of the mean. Analysis was performed using GraphPad Prism 6.0 (GraphPad Software, US, SCR 002798) and SPSS 19.0 (IBM, US, SCR 002865) software. Differences were analyzed using unpaired one-way ANOVA, and multiple comparisons were analyzed using Tukey’s test except brain water content, which was analyzed using unpaired two-way ANOVA with multiple comparisons analyzed using the Sidak test. All differences were considered statistically significant at a P-value < 0.05. 3. Results 3.1. Foxo1 expression at different time points and in brain areas after ICH The highest levels of Foxo1 expression at different time points and in different brain areas after ICH are shown in Fig. 1. Foxo1 expression peaked at 12 h ICH (P < 0.01), compared with sham, 1 h, 6 h, 24 h, 48 h, and 72 h ICH rats (Fig. 1A). Quantification of Foxo1 expression was shown in Fig. 1B. The highest level of Foxo1 expression and mRNA was in the ipsilateral corpus striatum (P < 0.01), compared with the ipsilateral cortex, or contralateral corpus striatum and cortex (Fig. 1C). The quantification and the relative mRNA level of Foxo1 expression was shown in Fig. 1D and E.
at 12 h post-ICH compared with the sham operation group, and also significantly decreased by knocking down Foxo1 with siRNA. The quantification and the relative mRNA level of Foxo1 expression was shown in Fig. 2B and C. Neurological symptoms significantly improved 12/24/72 h post-ICH and Garcia scores were significantly reduced (Fig. 2D). Brain water content was also significantly reduced 12/24/72 h post-ICH (Fig. 2E). In addition, ipsilateral brain water content increased compared with the contralateral side except in the sham operation rats (Fig. 2E). 3.3. Foxo1 knockdown decreased expression of downstream inflammation factors via Foxo1 pathways Expression of downstream inflammation factors is shown in Fig. 3. TLR4, NF-B, TNF-␣, IL-1, and IL-18 expression levels were significantly increased at 12 h post-ICH compared with the sham operation group, and were also significantly decreased by knocking down Foxo1 with siRNA, compared with the 12 h ICH and ICH + control siRNA groups (Fig. 3A). TLR4, NF-B, TNF-␣, IL-1, IL-18 quantifications were shown at Fig. 3B–F. 3.4. Foxo1 knockdown decreased MPO, IL-1ˇ, and IL-18 activity via Foxo1 pathways As shown in Fig. 4, MPO (Fig. 4A), IL-1 (Fig. 4B), and IL-18 (Fig. 4C) activity were significantly increased at 12 h post-ICH, compared with the sham operation group, and were also significantly decreased by knocking down Foxo1 with siRNA, compared with the 12 h ICH and ICH + control siRNA groups. 4. Discussion
3.2. Foxo1 knockdown decreased intracerebral hemorrhage injury and improved neurological function The effect of Foxo1 siRNA after ICH is shown in Fig. 2. Foxo1 expression levels (Fig. 2A) and mRNA were significantly increased
ICH is a potentially fatal condition with no effective clinical treatment. Although patients may survive the initial attack, the hematoma increases in size and may lead to a series of lifethreatening pathological physiological reactions, such as edema
134
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
Fig. 2. Decrease in intracerebral hemorrhage (ICH) injury and improved neurological function following knockdown of Foxo1. (A) Western blot assay for Foxo1 expression in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats. (B) Quantification of Foxo1 expression in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats (12 h ICH + siRNA vs. 12 h ICH and ICH + control siRNA group, **P < 0.01, n = 4 per group). (C) Quantitative RT-PCR assay for mRNA level of Foxo1 in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats; data were normalized to -actin gene (12 h ICH + siRNA vs. 12 h ICH and ICH + control siRNA group, **P < 0.01, n = 4 per group). (D) Garcia neurological score assay in sham, 12/24/72 h ICH, 12/24/72 h ICH + siRNA, 12/24/72 h ICH + control siRNA, (12/24/72 h ICH + siRNA vs. 12/24/72 h ICH and 12/24/72 h ICH + control siRNA group. **P < 0.01, n = 6 per group. (E) Brain water content assay in sham, 12/24/72 h ICH, 12/24/72 h ICH + siRNA, 12/24/72 h ICH + control siRNA, (12/24/72 h ICH + siRNA vs. 12/24/72 h ICH and 12/24/72 h ICH + control siRNA group and each ipsilateral side vs. its contralateral side. **P < 0.01, n = 4 per group.
Fig. 3. Decreased expression of downstream inflammation factors through Foxo1 pathways following Foxo1 knockdown. (A) Western blot assay for TLR4, NF-B, TNF-␣, IL-1, and IL-18 expression in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, and 12 h ICH + control siRNA rats. (B–F) Quantification of TLR4 (B), NF-B (C), TNF-␣ (D), IL-1 (E), and IL-18 (F) expression in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats (12 h ICH + siRNA vs. 12 h ICH and ICH + control siRNA group, **P < 0.01, n = 4 per group, data represented as mean ± standard error).
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
135
Fig. 4. Decreased MPO, IL-1, and IL-18 protein concentration and reduced inflammation factor activity following Foxo1 knockdown. (A–C) ELISA assay for MPO (A), IL-1 (B), and IL-18 (C) activity levels in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats (12 h ICH + siRNA vs. 12 h ICH and ICH + control siRNA group, **P < 0.01, n = 4 per group; data represented as mean ± standard error).
accumulation, inflammatory-cytokine release, and neurobehavioral deficits [2]. The 1-month mortality rate is 42%, and most survivors are left permanently disabled [15]. Current evidence shows that infiltrated neutrophils are observed at 4 h in the ICH rat model, leading to reactive oxygen species generation and proinflammatory protease expression. In addition, dying neutrophils promote inflammatory tissue injury by stimulating macrophages to release proinflammatory factors [16,17]. Toll-like receptors, as receptors for autoimmune activation, have been recognized as having a vital role in gram-negative bacteria infection and septic shock. Recent studies have shown that in subarachnoid hemorrhage, expression of TLR4 is increased and inflammatory factors are released, an important mechanism leading to hemorrhagic inflammation injury [18]. NF-B is a ubiquitous inducible transcription factor that promotes an inflammatory response and release of TNF-␣, IL-1, and IL-18. Moreover, NF-B is regulated by TLR4, an upstream factor [19]. TLR4/NF-B has been demonstrated as a fundamental inflammatory signaling pathway, with MPO related to neutrophil infiltration and improving edema formation. This suggests MPO is an important marker of inflammatory mechanisms after ICH [20]. All of these inflammatory factors are regulated by Foxo1, which begins the inflammatory reaction by regulating the TLR4/NF-B signaling pathway [10,11,21]. The Foxo family belongs to the forkhead box class of transcription factors. Foxo1 is expressed in numerous cell types and tissues, including endothelial cells, neural crest cells, smooth muscle cells, adipose tissue, trigeminal ganglia, and cardiomycoytes [22–25]. Previous research has shown that Foxo1 is expressed in brain tissue [8–10], but Foxo1 expression at different time points and in different brain areas after ICH was not yet clear. In our study, we found that Foxo1 expression was increased at 1 h following ICH, peaked around 12 h, and gradually decreased to 72 h, possibly in response to stress caused by surgery and ICH. This was similar to a previous study with patients with acute episode ICH and those in recovery [2]. In addition, we found that Foxo1 expression levels were highest in the ipsilateral corpus striatum, suggesting that an inflammatory response could produce a hemorrhagic stigma effect and be related to Foxo1 expression. It has been reported that losing Foxo1 could increase cell proliferation, resistance to apoptosis, and decrease inflammatory injury in Foxo1-knocked-out mice and Foxo1-knocked-down human pulmonary artery smooth muscle cells (PASMSs) [26]. In our study, Foxo1 knocked down by siRNA effectively inhibited Foxo1 expression and reduced Foxo1 mRNA levels. Neurological function and brain water content were also tested, and the results demonstrated that Foxo1 siRNA treatment could significantly increase neurological function and decrease brain water content in the ipsilateral hemisphere. These data suggest that abatement of Foxo1 is beneficial, and an elevation of Foxo1 harmful, after ICH. However, the mechanism of Foxo1-induced inflammation after ICH has not been studied.
Previous research has demonstrated that Foxo1 plays a crucial role in brain inflammation [9–11]. However, Foxo1 expression in brain tissue after ICH has not been reported. We now discuss the potential downstream signaling of Foxo1, which we hope will explain the mediation of inflammatory injury. As mentioned above, Foxo1 regulates many inflammatory factors such as TRL4, NF-B, TNF-␣, IL-1, and IL-18, via the TLR4/NF-B signaling pathway, which starts the inflammatory reaction [10,11,21]. Therefore, we hypothesized that Foxo1 was a major regulator of inflammatory injury following ICH. In our study, we found that Foxo1 knockdown by siRNA effectively inhibited expression of TRL4, NF-B, TNF-␣, IL1, and IL-18. These results reveal that, after ICH, Foxo1 is the key upstream factor of these inflammatory factors. It has been shown that blood-derived leukocytes and neutrophils infiltrate the brain parenchyma, worsening edema formation, and initiating an inflammatory reaction [3]. We found that Foxo1 knockdown by siRNA effectively decreased MPO activity. It was revealed that Foxo1 was related to leukocyte and neutrophil infiltration. To demonstrate the relationship between Foxo1 and inflammatory injury in more depth, we also tested the activity of IL-1 and IL-18, which are the most important downstream proinflammatory cytokines produced by activated macrophages, natural killer cells, and T-cell proliferation [5]. Results showed that Foxo1 knockdown by siRNA also decreased IL-1 and IL-18 activity, demonstrating that Foxo1 could regulate the activity of its downstream proinflammatory cytokines. In summary, our results showed that the mechanism of Foxo1-induced inflammatory injury was to regulate blood-derived leukocytes, neutrophil infiltration, activated macrophage accumulation, natural killer cell cytotoxicity, and T-cell proliferation via the TLR4/NF-B signaling pathway. In conclusion, our findings suggest that Foxo1 is a key transcription factor regulating ICH-triggered cerebral injury. Foxo1 knockdown may ameliorate brain edema, improve neurological function, and reduce neurological injury. Moreover, Foxo1 knockdown notably inhibited downstream inflammatory cytokine expression, decreasing the activity of these cytokines. The crucial role of Foxo1/TLR4/NF-B in the regulation of inflammatory lesions after ICH was also validated. In addition, we investigated peak Foxo1 expression levels after ICH, which were found at 12 h and within the ipsilateral corpus striatum. These data may provide a rationale for novel therapeutic approaches to manage local inflammatory responses in hemorrhagic stroke. 5. Conclusions We found that Foxo1 expression peaked at 12 h postintracerebral hemorrhage (ICH) and in the ipsilateral corpus striatum. Knockdown by Foxo1 siRNA decreased ICH injury, improved neurological function and decreased the expression of inflammatory factors downstream of the Foxo1 pathway. Our findings demonstrate that Foxo1 is a key regulator of inflammatory
136
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
injury in rats after ICH and provide a novel rationale for therapeutic approaches to managing inflammatory injury after ICH. Authors’ contributions All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Li zhenyu, You Yan, Zhao Yong, Li Lingyu and Zhao Jing. Acquisition of data: Li zhenyu, You Yan and He Qi. Analysis and interpretation of data:Li zhenyu, You Yan and Zhai Xuan. Drafting of the manuscript: Li zhenyu, You Yan and Li Lingyu. Critical revision of the manuscript for important: Li zhenyu, You Yan and Hou Yanghao. Statistical analysis: Li zhenyu, You Yan and He Faming. Obtained funding: Zhao Yong, Li Lingyu and Zhao Jing. Administrative, technical, and material support: Zhao Yong, Li Lingyu and Zhao Jing. Study supervision: Zhao Yong, Li Lingyu and Zhao Jing. Significant statement Foxo1, as the foremost member of the Foxo family, regulates inflammatory injury. We found that Foxo1 expression peaked at 12 h post-intracerebral hemorrhage (ICH) and in the ipsilateral corpus striatum. Knockdown by Foxo1 siRNA decreased ICH injury, improved neurological function, and decreased the expression of inflammatory factors downstream of the Foxo1 pathway. Foxo1 immunoregulation function in brain tissue after ICH in rats was uncertain, so our findings demonstrate that Foxo1 is a key regulator of inflammatory injury in rats after ICH. By identifying the molecular mechanisms of Foxo1/TLR4/NF-B signaling, we provide a novel rationale for therapeutic approaches to managing inflammatory injury after ICH. Acknowledgments This work was supported by The National Natural Science Foundation of China (No. 81171090 and 81271460), Natural Science Youth Foundation of China (No. 81301125) and the Medical scientific research projects of Chongqing (20120221). We would like to thank Dr. Leonard L. Seelig, Jr. for the excellent editorial support. References [1] J. Aronowski, et al., Molecular pathophysiology of cerebral hemorrhage: sec-ondary brain injury, Stroke 42 (2011) 1781–1786. [2] D. Strbian, et al., Rodent models of hemorrhagic stroke, Curr. Pharm. Des. 14 (2008) 352–358. [3] J. Wang, et al., Inflammation after intracerebral hemorrhage, J. Cereb. Blood Flow Metab. 27 (2007) 894–908.
[4] M. Qingyi, et al., NLRP3 inflammasome contributes to inflammation after intracerebral hemorrhage, Ann. Neurol. 75 (2014) 209–219. [5] F. Hsin Yuan, et al., Inducible heat shock protein 70, interleukin-18, and tumor necrosis factor alpha correlate with outcomes in spontaneous intracerebral hemorrhage, J. Clin. Neurosci. 14 (2007) 435–441. [6] Liang Feng, et al., P2X7R blockade prevents NLRP3 inflammasome activation and brain injury in a rat model of intracerebral hemorrhage: involvementof peroxynitrite, J. Neuroinflammation 12 (2015) 190. [7] K.C. Arden, FOXO animal models reveal a variety of diverse roles for FOXO transcription factors, Oncogene 27 (2008) 2345–2350. [8] K.K. Ho, et al., Many forks in the path: cycling with FoxO, Oncogene 27 (2008) 2300–2311. [9] Martha Lappas, Forkhead box O1 (Foxo1) in pregnant human myometrial cells: a role as a pro-inflammatory mediator in humanparturition, J. Reprod. Immunol. 99 (2013) 24–32. [10] Min Zhou, et al., PTEN-Foxo1 triggers innate inmune responses in acute leng injure, Immunol. Res. 62 (2015) 95–105. [11] Naoko Kamo, et al., PTEN-mediated AKT/-catenin/Foxo1 signaling regulates innate immune responses in mouse liver ischemia/reperfusion injury, Hepatology 57 (2013) 289–298. [12] J.H. Garcia, S. Wagner, et al., Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion inrats Statistical validation, Stroke 26 (4) (1995) 627–634. [13] L. Li, et al., Targeting Thioredoxin-1 with siRNA exacerbates oxidative stress injury after cerebral ischemia/reperfusion in rats, Neuroscience 284 (2015) 815–823. [14] M. Liu, et al., Etomidate mitigates pro-inflammatory cytokine production, Inflammation 17 (2015) 1–9. [15] V.L. Feigin, et al., Stroke epidemiology: a review of population-based studiesof incidence, prevalence, and case-fatality in the late 20th century, Lancet Neurol. 2 (2003) 43–53. [16] S.J. Weiss, Tissue destruction by neutrophils, N. Engl. J. Med. 320 (1989) 325–376. [17] M. Stern, et al., Human monocyte-derived macrophage phagocytosis of senescent eosinophils undergoing apoptosis. Mediation by alpha v beta 3/CD36/thrombospondin recognition mechanism and lack of phlogistic response, Am. J. Pathol. 149 (1996) 911–921. [18] A. Tsung, et al., The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion, J. Exp. Med. 201 (2005) 1135–1143. [19] Q. Li, et al., NF-kappaB regulation in the immune system, Nat. Rev. Immunol. 2 (2002) 725–734. [20] Elena Titova, et al., Reduced brain injury in CD18 deficient mice after experimental intracerebral hemorrhage, J. Neurosci. Res. 86 (2008) 3240–3245. [21] R.R. Daniela, et al., Interferon-gamma production by human neutrophils upon stimulation by IL-12, IL-15 and IL-18 and challenge with Paracoccidioides brasiliensis, Cytokine 69 (2014) 102–109. [22] T. Hosaka, et al., Disruption of forkhead transcription factor (FOXO) Family members in mice reveals their functional diversification, Proc. Natl. Acad. Sci. 101 (2004) 2975–2980. [23] T. Furuyama, et al., Abnormal angiogenesis in foxo1 (Fkhr)-deficient mice, J. Biol. Chem. 279 (2004) 34741–34749. [24] T. Furuyama, et al., Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues, Biochem. J. 349 (2000) 629–634. [25] H.J. Evans-Anderson, et al., Regulation of cardiomyocyte proliferation and myocardial growth during development by FOXO transcription factors, Circ. Res. 102 (2008) 686–694. [26] R. Savai, et al., Pro-proliferative and inflammatory signaling converge on Foxo1 transcription factor in pulmonary hypertension, Nat. Med. 20 (2014) 1289–1300.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Foxo1-mediated inflammatory response after cerebral hemorrhage in rats Li Zhenyu a , He Qi a , Zhai Xuan c , You Yan a , Li Lingyu b , Hou Yanghao a , He Faming d , Zhao Yong b , Zhao Jing a,∗ a
Department of Pathophysiology, Chongqing Medical University, Chongqing, People’s Republic of China Department of Pathology, Chongqing Medical University, Chongqing, People’s Republic of China c Department of Neurosurgery, The Children’s Hospital of Chongqing Medical University, Chongqing, People’s Republic of China d Department of Emergency, The First Affiliated Hospital of Chongqing Medical University. Chongqing, People’s Republic of China b
h i g h l i g h t s • • • • •
Foxo1 expression peaked at 12 h post-intracerebral hemorrhage (ICH) and in the ipsilateral corpus striatum. Intracerebroventricular injection of Foxo1 siRNA effectively inhibited Foxo1 mRNA and protein expression. Foxo1 siRNA significantly increased neurological function and decreased brain water content after ICH injury. Foxo1 siRNA obviously reduced inflammatory factors release. Inhibition of Foxo1 may inhibited the TLR4/NF-B pathway after ICH injury.
a r t i c l e
i n f o
Article history: Received 20 April 2016 Received in revised form 30 May 2016 Accepted 6 June 2016 Available online 7 June 2016 Keywords: Foxo1 Inflammatory injury TLR4 Immunoregulation Intracerebral hemorrhage MPO
a b s t r a c t The forkhead box O (Foxo) family of transcription factors plays a crucial role in cell apoptosis, immune regulation, and tissue development. Foxo1, as the foremost member of the Foxo family, regulates a wide range of molecular signals in many tissues, including tumor, liver, and brain. This study investigated Foxo1 expression at different time points and in different brain areas, and the role of Foxo1 in vivo in regulating inflammatory injury in a rat model of autologous blood-injected cerebral hemorrhage injury. We found that Foxo1 expression peaked at 12 h post-intracerebral hemorrhage (ICH) and in the ipsilateral corpus striatum. Foxo1 knockdown by Foxo1 siRNA decreased ICH injury, improved neurological function, and decreased the expression of inflammatory factors downstream of the Foxo1 pathway, including TLR4, NF-B, TNF-␣, IL-1, and IL-18. Foxo1 knockdown also decreased the expression and activity of myeloperoxidase, IL-1, and IL-18. In conclusion, our findings demonstrate that Foxo1 is a key regulator of inflammatory injury in rats after ICH. By identifying the molecular mechanisms of Foxo1/TLR4/NF-B signaling, we provide a novel rationale for therapeutic approaches to managing inflammatory injury after ICH. © 2016 Published by Elsevier Ireland Ltd.
1. Introduction
Abbreviations: Foxo1, the forkhead box O1; ICH, intracerebral hemorrhage; siRNA, small interfering RNA; TLR4, toll-like receptor 4; NF-B, nuclear factork-gene binding; TNF-␣, tumor necrosis factor; IL-1, interleukin-1; IL-18, interleukin-18; MPO, myeloperoxidase; PASMSs, pulmonary artery smooth muscle cells; RT-PCR, reverse transcription-polymerase chain reaction; ELISA, enzymelinked immunosorbent assay; WW, wet weight; DW, dry weight; Ipsi-CS, ipsilateral corpus striatum; Ipsi-CX, ipsilateral cortex; Cont-CS, contralateral corpus striatum; Cont-CX, contralateral cortex. ∗ Corresponding author at: Department of Pathophysiology, Institute of Neuroscience, Chongqing Medical University, Yixueyuan Road 1, Chongqing, 400016, People’s Republic of China. E-mail address: [email protected] (J. Zhao). http://dx.doi.org/10.1016/j.neulet.2016.06.013 0304-3940/© 2016 Published by Elsevier Ireland Ltd.
Intracerebral hemorrhage (ICH), an important clinical classification of stroke, induces neuronal apoptosis, formation of perihematomal edema, inflammatory lesions, neurological impairment, and energy metabolism disorder [1]. ICH remains a tremendous challenge for which there is no effective therapy at present [2]. Cerebral edema starts with an inflammatory reaction following introduction of a hematoma. In particular, blood-derived leukocytes and neutrophils infiltrate the brain parenchyma, disrupting the blood–brain barrier and causing edema formation as well as deterioration of neurobehavioral function [3]. These
132
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
responses are consequences of increased levels of myeloperoxidase (MPO), IL-1, and IL-18 [4–6]. Therefore, it is important to find a key upstream transcription factor in the inflammatory reaction. The forkhead box O (Foxo) family of transcription factors plays a crucial role in cell apoptosis, inflammation, and tissue development. Foxo1, a member of the Foxo family, regulates a wide range of molecular signals in many tissue types, such as tumor, liver, and brain [7,8]. Previous studies have suggested that under inflammatory conditions, Foxo1 expression is upregulated, which could induce the production of cultured macrophage inflammatory factors [9]. Immunoregulation of Foxo1 was demonstrated by enhancing TLR4-mediated inflammation in lung and liver tissue, among others, but its immunoregulation function in brain tissue after ICH in rats was uncertain [10,11]. In this study, we investigated the role of Foxo1 in ICH using an experimental ICH model with Foxo1 knockdown. We evaluated whether downregulated Foxo1 could reduce brain damage. Furthermore, we hypothesized that Foxo1 could regulate inflammatory injury via the TLR4/NF-B signaling pathway and modulate inflammatory factor activity. 2. Material and methods 2.1. Animals The animal study was approved by the Institutional Animal Care and Use Committee at Chongqing Medical University. Eightweek-old male Sprague–Dawley rats (weight 280–320 g, sourced from the Chongqing Medical Animal Experimentation Center) were housed in a temperature- and light-controlled environment under pathogen-free conditions, and provided unlimited access to food and water, 12/12 light/dark with humidity 60 ± 5% and 22 ± 3 ◦ C. All rats were randomly allocated to the following group: sham surgery group, ICH group, Foxo1 siRNA group and control siRNA of Foxo1 group in blind manner.
siRNAs of Foxo1-rat-1612 were 5 -GCCAAGACCCACAUAAUCATT-3 (sense) and 5 -UGAUUAUGUGGGUCUUGGCTT-3 (antisense). All siRNA was synthesized, purified, desalted, and shipped in the 2 -deprotected, duplexed form. 2.4. Injection of Foxo1-rat-siRNA Each tube of Foxo1-rat-siRNA was dissolved in 20 L DEPC, oscillated, centrifuged, and oscillated again, then infused into the right paracele at anterior–posterior 1 mm, mediolateral 2 mm, and dorsoventral 3.5 mm (8 L per rat), and retained for 15 min. The ICH model was established 24 h later. The stable negative control and scramble siRNA of Foxo1-rat-1612 were administered using the same procedure. 2.5. Brain water content assay ICH rats were decapitated under deep anesthesia, and the brains were immediately removed. Each brain was divided into two halves: hemorrhagic (ipsilateral) and contralateral. Each part was weighed on an electronic analytical balance to give the wet weight (WW) and dried at 100 ◦ C for 24 h in the oven to determine the dry weight (DW). Brain water content was then calculated as [(WW − DW)/WW] × 100%. 2.6. Garcia neurological score assay [12] ICH rats were given a score between 0 and 18 on the Garcia test, which consists of six sections: spontaneous activity, axial sensation, vibrissae proprioception, limb outstretching, lateral turning, and forelimb walking, with a possible score of 0–3 for each section (0 = worst, 3 = best). 2.7. Western blot
ICH was induced with a single infusion model of autologous blood (50 L). The rats were randomly assigned to an experimental group, anesthetized with 10% chloral hydrate (1 mL/100 g) by intraperitoneal injection, and stabilized prone in a stereotactic head frame. The autologous blood was collected from the arteria femoralis on the operation side and transferred into an anticoagulation syringe with a microinjection pump. The blood was infused into the right corpus striatum anterior-posterior 0.2 mm, mediolateral 3.0 mm, and dorsoventral 5.8 mm. An injection of 10 L blood was delivered 5.0 mm dorsoventral and retained for 2 min; the final 40 L blood was given as a total injection at dorsoventral 5.8 mm and retained for 15 min. Sham-operated rats were only given a needle insertion.
Amounts of 30 g of protein from each brain sample were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Monoclonal rabbit antirat Foxo1 (C29H4, Cell Signaling Technology, Danvers, USA, 1:1000, AB 2106495, Rabbit Anti-Foxo1), TLR4 (BS3489, Bioworld Technology, Minnesota, USA, 1:500, AB 1662746, Rabbit AntiTLR4), NF-B (sc-372, Santa Cruz Biotechnology, Texas, USA, 1:1000, AB 632037, Rabbit Anti-NF-B), TNF-␣ (BS1857, Bioworld Technology, Minnesota, USA, 1:1000, AB1662107, Rabbit AntiTNF-␣), IL-1 (BS3506, Bioworld Technology, Minnesota, USA, 1:500, AB 1661842, Rabbit Anti-IL-1), IL-18 (sc-7954, Santa Cruz Biotechnology, Texas, USA, 1:200, AB 1564060, Rabbit Anti-IL18), and -actin (13E5, Cell Signaling Technology, Danvers, USA, 1:1000, AB 2223169, Rabbit Anti--actin) were used. Relative protein quantities were determined by densitometry and expressed in absorbance units.
2.3. Preparation of siRNA
2.8. Quantitative RT-PCR
The siRNA against Foxo1 was designed using RNA oligochemical synthesis (Genepharma, Shanghai, China). Four Foxo1-rat-siRNA fragments were used: Foxo1-rat1285 (sense: 5 -GCAGACACCUUGCUAUUCATT-3 , antisense: 5 -UGAAUAGCAAGGUGUCUGCTT-3 ); Foxo1-rat-1434 (sense: 5 -GAGGAUUGAACCAGUAUAATT-3 , antisense: 5 -UUAUACUGGUUCAAUCCUCTT-3 ); Foxo1-rat-1612 (sense: 5 -CCAGGCACCUCAUAACAAATT-3 , antisense: 5 UUUGUUAUGAGGUGCCUGGTT-3 ); and stable negative contol 5 -UUCUCCGAACGUGUCACGUTT-3 , antisense: 5 (sense: ACGUGACACGUUCGGAGAATT-3 ). The most significant fragment screened by western blot was Foxo1-rat-1612, and the scramble
Total RNA was extracted from frozen brain using Reagent Kit(TaKaRa Biotechnology, Dalian, China). A total of 40 L RNA was reverse-transcribed into cDNA. Quantitative PCR was performed as described [13]. Primer sequences for the amplification of Foxo1 and -actin. Target gene expressions were calculated by their ratios to -actin.
2.2. ICH rat model
2.9. Enzyme-linked immunosorbent assay The presence of MPO was used as an index of neutrophil accumulation in the post-ICH brain tissue homogenate. The presence of IL-1 and IL-18 was used as an index of inflammatory injury in
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
133
Fig. 1. Foxo1 expression at different time points and brain areas after intracerebral hemorrhage (ICH). (A) Western blot assay for profiles of Foxo1 expression in sham, 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h ICH rats. (B) Quantification of Foxo1 expression in the ipsilateral corpus striatum in sham, 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h ICH rats. (12 h vs. other groups, **P < 0.01, n = 4 per group). (C) Western blot assay for profiles of Foxo1 expression in the ipsilateral corpus striatum and cortex, or contralateral corpus striatum and cortex in 12 h ICH. (D) Quantification of Foxo1 expression in the ipsilateral corpus striatum and cortex, or contralateral corpus striatum and cortex in 12 h ICH rats (ipsilateral corpus striatum vs. other groups, **P < 0.01, n = 4 per group). (E) Quantitative RT-PCR assay for mRNA level of Foxo1 in the ipsilateral corpus striatum and cortex, or contralateral corpus striatum and cortex, in 12 h ICH rats; data were normalized to -actin gene (ipsilateral corpus striatum vs. other groups, **P < 0.01, n = 4 per group; data represented as mean ± standard error).
the post-ICH brain tissue homogenate. MPO, IL-1, and IL-18 activity levels were assayed by ELISA kits(Boster Biological Technology, Wuhan, China) as previously described [14]. 2.10. Statistics Data were expressed as mean ± standard error of the mean. Analysis was performed using GraphPad Prism 6.0 (GraphPad Software, US, SCR 002798) and SPSS 19.0 (IBM, US, SCR 002865) software. Differences were analyzed using unpaired one-way ANOVA, and multiple comparisons were analyzed using Tukey’s test except brain water content, which was analyzed using unpaired two-way ANOVA with multiple comparisons analyzed using the Sidak test. All differences were considered statistically significant at a P-value < 0.05. 3. Results 3.1. Foxo1 expression at different time points and in brain areas after ICH The highest levels of Foxo1 expression at different time points and in different brain areas after ICH are shown in Fig. 1. Foxo1 expression peaked at 12 h ICH (P < 0.01), compared with sham, 1 h, 6 h, 24 h, 48 h, and 72 h ICH rats (Fig. 1A). Quantification of Foxo1 expression was shown in Fig. 1B. The highest level of Foxo1 expression and mRNA was in the ipsilateral corpus striatum (P < 0.01), compared with the ipsilateral cortex, or contralateral corpus striatum and cortex (Fig. 1C). The quantification and the relative mRNA level of Foxo1 expression was shown in Fig. 1D and E.
at 12 h post-ICH compared with the sham operation group, and also significantly decreased by knocking down Foxo1 with siRNA. The quantification and the relative mRNA level of Foxo1 expression was shown in Fig. 2B and C. Neurological symptoms significantly improved 12/24/72 h post-ICH and Garcia scores were significantly reduced (Fig. 2D). Brain water content was also significantly reduced 12/24/72 h post-ICH (Fig. 2E). In addition, ipsilateral brain water content increased compared with the contralateral side except in the sham operation rats (Fig. 2E). 3.3. Foxo1 knockdown decreased expression of downstream inflammation factors via Foxo1 pathways Expression of downstream inflammation factors is shown in Fig. 3. TLR4, NF-B, TNF-␣, IL-1, and IL-18 expression levels were significantly increased at 12 h post-ICH compared with the sham operation group, and were also significantly decreased by knocking down Foxo1 with siRNA, compared with the 12 h ICH and ICH + control siRNA groups (Fig. 3A). TLR4, NF-B, TNF-␣, IL-1, IL-18 quantifications were shown at Fig. 3B–F. 3.4. Foxo1 knockdown decreased MPO, IL-1ˇ, and IL-18 activity via Foxo1 pathways As shown in Fig. 4, MPO (Fig. 4A), IL-1 (Fig. 4B), and IL-18 (Fig. 4C) activity were significantly increased at 12 h post-ICH, compared with the sham operation group, and were also significantly decreased by knocking down Foxo1 with siRNA, compared with the 12 h ICH and ICH + control siRNA groups. 4. Discussion
3.2. Foxo1 knockdown decreased intracerebral hemorrhage injury and improved neurological function The effect of Foxo1 siRNA after ICH is shown in Fig. 2. Foxo1 expression levels (Fig. 2A) and mRNA were significantly increased
ICH is a potentially fatal condition with no effective clinical treatment. Although patients may survive the initial attack, the hematoma increases in size and may lead to a series of lifethreatening pathological physiological reactions, such as edema
134
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
Fig. 2. Decrease in intracerebral hemorrhage (ICH) injury and improved neurological function following knockdown of Foxo1. (A) Western blot assay for Foxo1 expression in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats. (B) Quantification of Foxo1 expression in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats (12 h ICH + siRNA vs. 12 h ICH and ICH + control siRNA group, **P < 0.01, n = 4 per group). (C) Quantitative RT-PCR assay for mRNA level of Foxo1 in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats; data were normalized to -actin gene (12 h ICH + siRNA vs. 12 h ICH and ICH + control siRNA group, **P < 0.01, n = 4 per group). (D) Garcia neurological score assay in sham, 12/24/72 h ICH, 12/24/72 h ICH + siRNA, 12/24/72 h ICH + control siRNA, (12/24/72 h ICH + siRNA vs. 12/24/72 h ICH and 12/24/72 h ICH + control siRNA group. **P < 0.01, n = 6 per group. (E) Brain water content assay in sham, 12/24/72 h ICH, 12/24/72 h ICH + siRNA, 12/24/72 h ICH + control siRNA, (12/24/72 h ICH + siRNA vs. 12/24/72 h ICH and 12/24/72 h ICH + control siRNA group and each ipsilateral side vs. its contralateral side. **P < 0.01, n = 4 per group.
Fig. 3. Decreased expression of downstream inflammation factors through Foxo1 pathways following Foxo1 knockdown. (A) Western blot assay for TLR4, NF-B, TNF-␣, IL-1, and IL-18 expression in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, and 12 h ICH + control siRNA rats. (B–F) Quantification of TLR4 (B), NF-B (C), TNF-␣ (D), IL-1 (E), and IL-18 (F) expression in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats (12 h ICH + siRNA vs. 12 h ICH and ICH + control siRNA group, **P < 0.01, n = 4 per group, data represented as mean ± standard error).
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
135
Fig. 4. Decreased MPO, IL-1, and IL-18 protein concentration and reduced inflammation factor activity following Foxo1 knockdown. (A–C) ELISA assay for MPO (A), IL-1 (B), and IL-18 (C) activity levels in the ipsilateral corpus striatum in sham, 12 h ICH, 12 h ICH + siRNA, 12 h ICH + control siRNA rats (12 h ICH + siRNA vs. 12 h ICH and ICH + control siRNA group, **P < 0.01, n = 4 per group; data represented as mean ± standard error).
accumulation, inflammatory-cytokine release, and neurobehavioral deficits [2]. The 1-month mortality rate is 42%, and most survivors are left permanently disabled [15]. Current evidence shows that infiltrated neutrophils are observed at 4 h in the ICH rat model, leading to reactive oxygen species generation and proinflammatory protease expression. In addition, dying neutrophils promote inflammatory tissue injury by stimulating macrophages to release proinflammatory factors [16,17]. Toll-like receptors, as receptors for autoimmune activation, have been recognized as having a vital role in gram-negative bacteria infection and septic shock. Recent studies have shown that in subarachnoid hemorrhage, expression of TLR4 is increased and inflammatory factors are released, an important mechanism leading to hemorrhagic inflammation injury [18]. NF-B is a ubiquitous inducible transcription factor that promotes an inflammatory response and release of TNF-␣, IL-1, and IL-18. Moreover, NF-B is regulated by TLR4, an upstream factor [19]. TLR4/NF-B has been demonstrated as a fundamental inflammatory signaling pathway, with MPO related to neutrophil infiltration and improving edema formation. This suggests MPO is an important marker of inflammatory mechanisms after ICH [20]. All of these inflammatory factors are regulated by Foxo1, which begins the inflammatory reaction by regulating the TLR4/NF-B signaling pathway [10,11,21]. The Foxo family belongs to the forkhead box class of transcription factors. Foxo1 is expressed in numerous cell types and tissues, including endothelial cells, neural crest cells, smooth muscle cells, adipose tissue, trigeminal ganglia, and cardiomycoytes [22–25]. Previous research has shown that Foxo1 is expressed in brain tissue [8–10], but Foxo1 expression at different time points and in different brain areas after ICH was not yet clear. In our study, we found that Foxo1 expression was increased at 1 h following ICH, peaked around 12 h, and gradually decreased to 72 h, possibly in response to stress caused by surgery and ICH. This was similar to a previous study with patients with acute episode ICH and those in recovery [2]. In addition, we found that Foxo1 expression levels were highest in the ipsilateral corpus striatum, suggesting that an inflammatory response could produce a hemorrhagic stigma effect and be related to Foxo1 expression. It has been reported that losing Foxo1 could increase cell proliferation, resistance to apoptosis, and decrease inflammatory injury in Foxo1-knocked-out mice and Foxo1-knocked-down human pulmonary artery smooth muscle cells (PASMSs) [26]. In our study, Foxo1 knocked down by siRNA effectively inhibited Foxo1 expression and reduced Foxo1 mRNA levels. Neurological function and brain water content were also tested, and the results demonstrated that Foxo1 siRNA treatment could significantly increase neurological function and decrease brain water content in the ipsilateral hemisphere. These data suggest that abatement of Foxo1 is beneficial, and an elevation of Foxo1 harmful, after ICH. However, the mechanism of Foxo1-induced inflammation after ICH has not been studied.
Previous research has demonstrated that Foxo1 plays a crucial role in brain inflammation [9–11]. However, Foxo1 expression in brain tissue after ICH has not been reported. We now discuss the potential downstream signaling of Foxo1, which we hope will explain the mediation of inflammatory injury. As mentioned above, Foxo1 regulates many inflammatory factors such as TRL4, NF-B, TNF-␣, IL-1, and IL-18, via the TLR4/NF-B signaling pathway, which starts the inflammatory reaction [10,11,21]. Therefore, we hypothesized that Foxo1 was a major regulator of inflammatory injury following ICH. In our study, we found that Foxo1 knockdown by siRNA effectively inhibited expression of TRL4, NF-B, TNF-␣, IL1, and IL-18. These results reveal that, after ICH, Foxo1 is the key upstream factor of these inflammatory factors. It has been shown that blood-derived leukocytes and neutrophils infiltrate the brain parenchyma, worsening edema formation, and initiating an inflammatory reaction [3]. We found that Foxo1 knockdown by siRNA effectively decreased MPO activity. It was revealed that Foxo1 was related to leukocyte and neutrophil infiltration. To demonstrate the relationship between Foxo1 and inflammatory injury in more depth, we also tested the activity of IL-1 and IL-18, which are the most important downstream proinflammatory cytokines produced by activated macrophages, natural killer cells, and T-cell proliferation [5]. Results showed that Foxo1 knockdown by siRNA also decreased IL-1 and IL-18 activity, demonstrating that Foxo1 could regulate the activity of its downstream proinflammatory cytokines. In summary, our results showed that the mechanism of Foxo1-induced inflammatory injury was to regulate blood-derived leukocytes, neutrophil infiltration, activated macrophage accumulation, natural killer cell cytotoxicity, and T-cell proliferation via the TLR4/NF-B signaling pathway. In conclusion, our findings suggest that Foxo1 is a key transcription factor regulating ICH-triggered cerebral injury. Foxo1 knockdown may ameliorate brain edema, improve neurological function, and reduce neurological injury. Moreover, Foxo1 knockdown notably inhibited downstream inflammatory cytokine expression, decreasing the activity of these cytokines. The crucial role of Foxo1/TLR4/NF-B in the regulation of inflammatory lesions after ICH was also validated. In addition, we investigated peak Foxo1 expression levels after ICH, which were found at 12 h and within the ipsilateral corpus striatum. These data may provide a rationale for novel therapeutic approaches to manage local inflammatory responses in hemorrhagic stroke. 5. Conclusions We found that Foxo1 expression peaked at 12 h postintracerebral hemorrhage (ICH) and in the ipsilateral corpus striatum. Knockdown by Foxo1 siRNA decreased ICH injury, improved neurological function and decreased the expression of inflammatory factors downstream of the Foxo1 pathway. Our findings demonstrate that Foxo1 is a key regulator of inflammatory
136
Z. Li et al. / Neuroscience Letters 629 (2016) 131–136
injury in rats after ICH and provide a novel rationale for therapeutic approaches to managing inflammatory injury after ICH. Authors’ contributions All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Li zhenyu, You Yan, Zhao Yong, Li Lingyu and Zhao Jing. Acquisition of data: Li zhenyu, You Yan and He Qi. Analysis and interpretation of data:Li zhenyu, You Yan and Zhai Xuan. Drafting of the manuscript: Li zhenyu, You Yan and Li Lingyu. Critical revision of the manuscript for important: Li zhenyu, You Yan and Hou Yanghao. Statistical analysis: Li zhenyu, You Yan and He Faming. Obtained funding: Zhao Yong, Li Lingyu and Zhao Jing. Administrative, technical, and material support: Zhao Yong, Li Lingyu and Zhao Jing. Study supervision: Zhao Yong, Li Lingyu and Zhao Jing. Significant statement Foxo1, as the foremost member of the Foxo family, regulates inflammatory injury. We found that Foxo1 expression peaked at 12 h post-intracerebral hemorrhage (ICH) and in the ipsilateral corpus striatum. Knockdown by Foxo1 siRNA decreased ICH injury, improved neurological function, and decreased the expression of inflammatory factors downstream of the Foxo1 pathway. Foxo1 immunoregulation function in brain tissue after ICH in rats was uncertain, so our findings demonstrate that Foxo1 is a key regulator of inflammatory injury in rats after ICH. By identifying the molecular mechanisms of Foxo1/TLR4/NF-B signaling, we provide a novel rationale for therapeutic approaches to managing inflammatory injury after ICH. Acknowledgments This work was supported by The National Natural Science Foundation of China (No. 81171090 and 81271460), Natural Science Youth Foundation of China (No. 81301125) and the Medical scientific research projects of Chongqing (20120221). We would like to thank Dr. Leonard L. Seelig, Jr. for the excellent editorial support. References [1] J. Aronowski, et al., Molecular pathophysiology of cerebral hemorrhage: sec-ondary brain injury, Stroke 42 (2011) 1781–1786. [2] D. Strbian, et al., Rodent models of hemorrhagic stroke, Curr. Pharm. Des. 14 (2008) 352–358. [3] J. Wang, et al., Inflammation after intracerebral hemorrhage, J. Cereb. Blood Flow Metab. 27 (2007) 894–908.
[4] M. Qingyi, et al., NLRP3 inflammasome contributes to inflammation after intracerebral hemorrhage, Ann. Neurol. 75 (2014) 209–219. [5] F. Hsin Yuan, et al., Inducible heat shock protein 70, interleukin-18, and tumor necrosis factor alpha correlate with outcomes in spontaneous intracerebral hemorrhage, J. Clin. Neurosci. 14 (2007) 435–441. [6] Liang Feng, et al., P2X7R blockade prevents NLRP3 inflammasome activation and brain injury in a rat model of intracerebral hemorrhage: involvementof peroxynitrite, J. Neuroinflammation 12 (2015) 190. [7] K.C. Arden, FOXO animal models reveal a variety of diverse roles for FOXO transcription factors, Oncogene 27 (2008) 2345–2350. [8] K.K. Ho, et al., Many forks in the path: cycling with FoxO, Oncogene 27 (2008) 2300–2311. [9] Martha Lappas, Forkhead box O1 (Foxo1) in pregnant human myometrial cells: a role as a pro-inflammatory mediator in humanparturition, J. Reprod. Immunol. 99 (2013) 24–32. [10] Min Zhou, et al., PTEN-Foxo1 triggers innate inmune responses in acute leng injure, Immunol. Res. 62 (2015) 95–105. [11] Naoko Kamo, et al., PTEN-mediated AKT/-catenin/Foxo1 signaling regulates innate immune responses in mouse liver ischemia/reperfusion injury, Hepatology 57 (2013) 289–298. [12] J.H. Garcia, S. Wagner, et al., Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion inrats Statistical validation, Stroke 26 (4) (1995) 627–634. [13] L. Li, et al., Targeting Thioredoxin-1 with siRNA exacerbates oxidative stress injury after cerebral ischemia/reperfusion in rats, Neuroscience 284 (2015) 815–823. [14] M. Liu, et al., Etomidate mitigates pro-inflammatory cytokine production, Inflammation 17 (2015) 1–9. [15] V.L. Feigin, et al., Stroke epidemiology: a review of population-based studiesof incidence, prevalence, and case-fatality in the late 20th century, Lancet Neurol. 2 (2003) 43–53. [16] S.J. Weiss, Tissue destruction by neutrophils, N. Engl. J. Med. 320 (1989) 325–376. [17] M. Stern, et al., Human monocyte-derived macrophage phagocytosis of senescent eosinophils undergoing apoptosis. Mediation by alpha v beta 3/CD36/thrombospondin recognition mechanism and lack of phlogistic response, Am. J. Pathol. 149 (1996) 911–921. [18] A. Tsung, et al., The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion, J. Exp. Med. 201 (2005) 1135–1143. [19] Q. Li, et al., NF-kappaB regulation in the immune system, Nat. Rev. Immunol. 2 (2002) 725–734. [20] Elena Titova, et al., Reduced brain injury in CD18 deficient mice after experimental intracerebral hemorrhage, J. Neurosci. Res. 86 (2008) 3240–3245. [21] R.R. Daniela, et al., Interferon-gamma production by human neutrophils upon stimulation by IL-12, IL-15 and IL-18 and challenge with Paracoccidioides brasiliensis, Cytokine 69 (2014) 102–109. [22] T. Hosaka, et al., Disruption of forkhead transcription factor (FOXO) Family members in mice reveals their functional diversification, Proc. Natl. Acad. Sci. 101 (2004) 2975–2980. [23] T. Furuyama, et al., Abnormal angiogenesis in foxo1 (Fkhr)-deficient mice, J. Biol. Chem. 279 (2004) 34741–34749. [24] T. Furuyama, et al., Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues, Biochem. J. 349 (2000) 629–634. [25] H.J. Evans-Anderson, et al., Regulation of cardiomyocyte proliferation and myocardial growth during development by FOXO transcription factors, Circ. Res. 102 (2008) 686–694. [26] R. Savai, et al., Pro-proliferative and inflammatory signaling converge on Foxo1 transcription factor in pulmonary hypertension, Nat. Med. 20 (2014) 1289–1300.