- Email: [email protected]
Protein arginine methyltransferase-1 deficiency restrains depression-like behavior of mice by inhibiting inflammation and oxidative stress via Nrf-2
Biochemical and Biophysical Research Communications 518 (2019) 430e437
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Protein arginine methyltransferase-1 deficiency restrains depressionlike behavior of mice by inhibiting inflammation and oxidative stress via Nrf-2 Hongbo Liu, Jinghui Jiang, Lili Zhao* Department of Paediatrics, Liaocheng People's Hospital, Liaocheng, 252000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 July 2019 Accepted 7 August 2019 Available online 3 September 2019
Current evidence indicates that depression is accompanied by the activation of inflammatory response and oxidative stress. Protein arginine methyltransferase 1 (PRMT1) is a histone methyltransferase that methylates Arg3 on histone H4, playing crucial role in regulating various pathological processes. In the study, we attempted to explore the effects of PRMT1 on animal model with depression through a single administration of lipopolysaccharide (LPS). Our results indicated that PRMT1 knockout (PRMT1/) improved LPS-induced anxiety- and depressive-like behavior, along with up-regulated expression levels of brain-derived neurotrophic factor (BDNF) and PSD-95. Furthermore, PRMT1 deficiency significantly improved LPS-induced changes in dendritic spine density in the areas of prefrontal cortex (PFC), CA3 and dentate gyrus (DG), and nucleus accumbens (NAc). In addition, PRMT1 deletion ameliorated the neuroinflammatory responses, as evidenced by the reduced expression of interleukin 1b (IL-1b) and tumor necrosis factor (TNF)-a, which might be through repressing nuclear factor-kB (NF-kB) signaling. Moreover, oxidative stress induced by LPS was alleviated by PRMT1 knockout in hippocampus of mice at least partly via promoting Nrf-2 expressions. The anti-depressant effects of PRMT1 inhibition were verified in LPS-incubated astrocytes. Importantly, we found that PRMT1 knockout-alleviated inflammation and oxidative stress triggered by LPS were significantly recovered by the suppression of Nrf-2. Therefore, Nrf2 was markedly involved in PRMT1-regulated depression-like behavior. Taken together, the results indicated that PRMT1 might be an important therapeutic target for developing effective treatment to prevent depressive-like behavior. © 2019 Published by Elsevier Inc.
Keywords: Depression PRMT1 Inflammation Oxidative stress Nrf-2
1. Introduction Depression is one of the most common psychiatric disorders worldwide [1]. Despite the precise molecular mechanism underlying the pathophysiology of depression is unclear, multiple lines of evidence illustrate that inflammatory response and oxidative stress play a critical role in the pathophysiology of depression [2,3]. Also, a meta-analysis indicates higher blood levels of pro-inflammatory cytokines in drug-free depressed patients when compared to healthy controls [4,5]. Presently, peripheral administration of the bacterial endotoxin, LPS, triggers depression-like behavior in mice after the induction of inflammation, as well as oxidative stress [6]. Therefore, finding significant target associated with inflammation and oxidative stress might be effective for developing therapeutic strategy against depressive symptoms.
* Corresponding author. E-mail address: [email protected] (L. Zhao). https://doi.org/10.1016/j.bbrc.2019.08.032 0006-291X/© 2019 Published by Elsevier Inc.
Protein arginine methyltransferase-1 (PRMT1), methylating both histones and key cellular proteins, has been served as a crucial modulator of various cellular processes, such as signal transduction, protein and protein interaction, as well as transcriptional modulation [7,8]. For instance, PRMT1 played an essential role in gene activation and metamorphosis triggered by ligand activated thyroid hormone receptor [9]. In addition, PRMT1 expression participates in fibroblast proliferation and chronic airway inflammation in Aginduced pulmonary inflammation through the regulation of remodeling associated cytokines [10]. In vitro studies also suggested that angiotensin-II markedly induced PRMT1 expression and endothelial cell activation, thus producing reactive oxygen species (ROS) [11]. Thus, PRMT1 is involved in the meditation of inflammatory response and ROS generation. However, its influences on anxiety- and depression-like behaviors have not been investigated and reported. The purpose of the study is to explore if PRMT1 could affect LPSinduced animal model with depression. The results suggested that
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
PRMT1 deletion significantly improved anxiety- and depressionlike behaviors induced by LPS in mice. Inflammatory response and oxidative stress induced by LPS were obviously ameliorated by the knockout of PRMT1 in hippocampus of mice. Notably, the in vitro experiments using LPS-treated astrocytes demonstrated that PRMT1-meditated inflammatory and oxidative damage was largely dependent on Nrf-2. Together, our study elucidated that PRMT1 could be served as a therapeutic candidate to develop effective treatment for alleviating depression. 2. Materials and methods 2.1. Animals and treatments Wild type C57BL/6 mice (adult male, PRMT1þ/þ) weighing 22e25 g were obtained from the Vital River Laboratories (Beijing, China). PRMT1 knockout (PRMT1/) mice with C57BL/6 background were constructed and purchased from Model Animal Research Center of Nanjing University (Nanjing, China). All mice were housed in a 12-h dark/light cycle, temperature (20 ± 2 C) and humidity controlled environment with free access to water and food. 0.5 mg/kg of LPS (L-4130, serotype 0111:B4, Sigma-Aldrich, USA) was dissolved in distilled water. Saline (10 ml/kg) or LPS (0.5 mg/kg) was administered intraperitoneally (i.p.) to mice. Then behavior was measured following saline or LPS administration for 24 h. The experimental procedure was approved by the Animal Care and Use Committee of the First Affiliated Hospital of Harbin Medical University (Harbin, China). All efforts were made to reduce the number of animal used and their suffering. 2.2. Cells and incubation Astrocytes (AST) from PRMT1/ and PRMT1þ/þ mice were prepared essentially as described in the previous reports [12]. AST was then collected and seeded in DMEM/F12 medium (GIBCO Corporation, Gaithersburg, MD, USA), supplemented with 10% FBS (GIBCO Corporation), 1 105 U/L streptomycin sulfate with a concentration 1 106/mL at 37 C with 5% CO2. LPS and/or ML385 MedChem Express (Monmouth Junction, NJ, USA) were exposed to AST for further study.
431
homogenized in ice-cold RIPA buffer with Complete Protease Inhibitor (Roche, USA). The dissolved proteins were harvested after centrifugation at 12,000 g for 15 min at 4 C. Then, the supernatant was collected. A quantity of 25e50 mg total proteins was loaded onto a 10e12% SDS-PAGE, electrophoretically transferred to polyvinylidene difluoride membrane (PVDF) and probed with the following primary antibodies: phosphoeNFekB p65 (1:1000, Cell Signaling), NF-kB (1:1000, Cell Signaling). Nrf-2 (1:1000, Abcam, USA), GFAP (1:1000, Abcam), PRMT1 (1:1000, Abcam), and Iba-1 (1:1000, Santa Cruz Biotechnology, Inc.). GAPDH (1:1000, Abcam) was used as an internal control. Secondary antibodies were horseradish peroxidase (HRP) conjugated to goat/mouse antirabbit IgG (1:6000, Sigma Aldrich). The membranes were developed using an enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology). 2.6. Biochemical measurements Concentrations of MDA (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), SOD (Nanjing Jiancheng Bioengineering Institute), GSH-pX (Nanjing Jiancheng Bioengineering Institute), TNF-a (R&D System, USA) and IL-1b (R&D System) were quantified using commercial kits according to the manufacturer's protocol. 2.7. Immunofluorescent analysis The brain and AST cells were fixed with 4% paraformaldehyde. Then, the brain tissue was treated with 30% sucrose at 4 C for 48 h. Brain tissue was sectioned with a thickness of 10 mm and pasted into microscope slides, followed by blocking with buffer (BSA, PBS with 0.3% Triton X-100) for 1 h. The sections were incubated with anti-NeuN (1:200, Abcam), GFAP (1:200, Abcam), phosphoeNFekB p65 (1:200, Cell Signaling) or Nrf-2 (Abcam) overnight at 4 C. The sections were rinsed with PBS and incubated with fluorescent secondary antibodies (1:500, Invitrogen), for 1 h at room temperature. Nuclei were stained with DAPI (Solarbio, Beijing, China) for 15 min, mounted onto slides and coverslips with ProLong mounting medium (Invitrogen). The representative images were captured using a fluorescent microscope. 2.8. DCF-DA analysis
2.3. Behavior analysis Behavioral tests, including locomotion, tail suspension test (TST), forced swimming test, sucrose preference test and open field test, were performed as previously reported [13e17]. All behavioral tests were conducted during the illuminated part of the cycle (between 9:00 and 12:00 a.m.). Behavior experiments were monitored by a trained observer blind to treatment. 2.4. Real time-quantitative PCR (RT-qPCR) analysis Total RNA was extracted from the hippocampus and cells using Trizol reagent (Gibco) according to the manufacturer's instructions. Identical amounts of RNA were reversely transcribed into cDNA with a commercial RT-PCR kit (Fermentas, Vilnius, Lithuania) according to the manufacturer's instructions. Real-time RT-PCR was carried out with 1 ml cDNA on real-time system (Analtik Jena. AG. Germany) using an UltraSYBR mixture (CoWin Bioscience Co., Beijing, China) and normalized to b-actin. Protocol of the real-time PCR was performed. The primers were listed in Supplementary table 1. 2.5. Western blot analysis Frozen tissue from the entire hippocampus and cells was
ROS levels in AST were determined using the specific probe DCFDA (Life Technologies, Invitrogen, USA) following the manufacturer's instructions. The stained AST cells were analyzed. 2.9. Immunohistochemical analysis Brain tissues were cut with 5 mm thick coronal sections on a microtome, then de-waxed with xylene and rehydrated with ethanol. After washing with PBS, all sections were stained with 0.1% Cresyl violet (Sigma Aldrich, USA) for 30 min at 37 C. The representative images were captured using a light microscope for surviving nerve cells in hippocampus area. 2.10. Golgi staining Golgi staining was carried out using the FD Rapid GolgiStain™ Kit (FD Neuro Technologies, Inc., Columbia, USA), following the manufacturer's instructions as previously described [14,18,19]. Representative images of dendrites within CA1, CA3, and DG of the hippocampus, prelimbic and inflalimbic areas of medial prefrontal cortex (mPFC), and shell and core of nucleus accumbens (NAc) were captured with a Keyence BZ-9000 GenerationIImicroscope (Osaka, Japan). Spines were analyzed along CA1, CA3, DG, prelimbic and
432
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
inflalimbic areas of mPFC, as well as shell and core of NAc dendrites as previously indicated [18,20]. 2.11. Statistics Data were presented as the mean ± S.E.M. Statistical analysis of data was carried out by one-way analysis of variance (ANOVA), followed by post-hoc Tukey test. Differences were considered statistically significant if the p value was P < 0.05. 3. Results 3.1. The influence of PRMT1 on anxiety- and depression-like behaviors induced by LPS in mice In the regard, the effects of PRMT1 on anxiety- and depressionlike behaviors induced by LPS were investigated. In the locomotion
test, no significant differences were observed (Fig. 1A). TST and FST results suggested that PRMT1 knockout significantly attenuated the increased immobility time in mice after LPS treatment (Fig. 1B and C). Moreover, PRMT1 deletion produced a complementary upregulation in swimming time compared to LPS/PRMT1þ/þ group (Fig. 1D). In the sucrose preference test, LPS-challenged PRMT1þ/þ mice showed a marked decrease in sucrose preference index as compared to saline-treated mice, whereas PRMT1 knockout significantly improved the sucrose preference index in LPS-treated mice (Fig. 1E). Open field test indicated that the decrease in line crossings produced by LPS was markedly prevented by PRMT1 ablation (Fig. 1F). Moreover, the reduction in recognition index induced by LPS was significantly improved by PRMT1 knockout (Fig. 1G). As shown in Fig. 1H and I, the duration in the open arms and entries into the open arms reduced by LPS administration were improved in PRMT1/ mice. BDNF and PSD-95 are the prominent regulators of neuronal survival, growth, and differentiation during
Fig. 1. The influence of PRMT1 on anxiety- and depression-like behaviors induced by LPS in mice. (A) Locomotion (LST), (B) tail-suspension test (TST), (C) and forced swim test (FST) were performed 2 h, 4 h, and 6 h after 24 h of LPS administration. (D) Swimming time in each group of mice was measured. (E) Percentage of sucrose consumption in the sucrose preference test (SPT). (F) The number of crossing in open field test. (G) Recognition index for novel objects induced by LPS in mice. (H) Duration in the open arms. (I) The entries into the open arms. RT-qPCR analysis of (J) BDNF and (K) PSD-95 in brain areas of PFC, NAc, CA1, CA3 and DG in mice. Data were expressed as the mean ± S.E.M (n ¼ 15 per group for behavior tests and n ¼ 8 for RT-qPCR analysis). #P < 0.05 and ##P < 0.01 versus Con/PRMTþ/þ group; þP < 0.05 and þþP < 0.01 versus LPS/PRMTþ/þ group.
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
development [21,22]. As described in Fig. 1J and K, the expression levels of BDNF and PSD-95 were markedly decreased by LPS challenge in brain areas of PFC, NAc, CA3 and DG, which were reversed by the loss of PRMT1. The levels of BDNF and PSD-95 were not affected by LPS or PRMT1 deletion in CA1 area. The results above demonstrated that PRMT1 was involved in anxiety- and depression-like behaviors induced by LPS in mice.
433
dendritic spine density in shell and core of NAc in PRMT1þ/þ mice; however, the changes induced by LPS were reversed by PRMT1 deletion (Fig. 2A and B). The number of surviving cells was obviously reduced in hippocampus of LPS/PRMT1þ/þ mice, while being markedly alleviated in mice lacking of PRMT1 expression (Fig. 2C and D). RT-qPCR and western blot results indicated that LPSpromoted expression levels of GFAP and Iba-1 were clearly downregulated in PRMT1/ mice (Fig. 2E and F).
3.2. PRMT1 deletion alleviates brain injury of LPS-induced mice Golgi staining data suggested that LPS significantly reduced dendritic spine density in the prelimbic and infralimbic regions of PFC, CA3 and DG, but not in the CA1, of hippocampus, and enhanced
3.3. Knockout of PRMT1 attenuates LPS-induced inflammation and oxidative stress in hippocampus of mice The levels of IL-1b and TNF-a were significantly increased after
Fig. 2. PRMT1 deletion alleviates brain injury of LPS-induced mice. (A&B) Golgi staining in the brain regions, including prelimbic and infralimbic regions of PFC, NAc core and shell, CA1, CA3 and DG of hippocampus) was performed. Representative images and data of Golgi staining in the brain regions of mice. (C) Cresyl violet staining of CA1 area in hippocampus of mice. (D) Immunofluorescent analysis of NeuN in CA1 area of hippocampus. (E&F) RT-qPCR and western blot analysis of GFAP and Iba-1 in hippocampus of mice. Data were expressed as the mean ± S.E.M (n ¼ 8 per group). #P < 0.05, ##P < 0.01 and ###P < 0.001 versus Con/PRMTþ/þ group; þP < 0.05 and þþP < 0.01 versus LPS/PRMTþ/þ group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
434
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
24 h of LPS administration both in the hippocampus and PFC compared with those in control group of PRMT1þ/þ mice. Meanwhile, PRMT1 deficiency evidently decreased IL-1b and TNF-ain the hippocampus and PFC in LPS-challenged mice using RT-qPCR and ELISA analysis (Fig. 3AeD). In mice treated with LPS, phosphoeNFekB expressions were markedly increased in the hippocampus, whereas PRMT1 knockout showed significant inhibitory effect on phosphoeNFekB expression (Fig. 3E and F). The significant increase of MDA in hippocampus induced by LPS was markedly prevented by the loss of PRMT1 (Fig. 3G). In contrast, LPSsuppressed SOD and GSH-pX activities in hippocampus of mice were significantly restored by PRMT1 deletion (Fig. 3H and I). Immunofluorescent analysis suggested that Nrf-2 expression levels were markedly reduced by LPS challenge in hippocampus of mice, whereas being improved in PRMT1/ mice (Fig. 3J).
Nrf-2 on PRMT1-regulated central injury were explored considering the essential role of Nrf-2 in the meditation of inflammatory response and oxidative stress [23]. Next, Nrf-2 expression in AST was repressed by adding ML385, a significant Nrf-2 inhibitor (Fig. 4I). Importantly, PRMT1 knockout-reduced expression levels of IL-1b and TNF-a in LPS-exposed AST were regained by ML385 pre-treatment (Fig. 4J). Also, ML385 pre-treatment markedly recovered the expression of phosphoeNFekB in LPS-treated AST in the absence of PRMT1 (Fig. 4K). Furthermore, PRMT1/-suppressed ROS generation in LPS-exposed cells was markedly restored by ML385 pre-treatment (Fig. 4L). The results above indicated that PRMT1-regulated inflammation and oxidative stress in LPS-treated astrocytes were partly dependent on Nrf-2, thereby modulating anxiety- and depression-like behaviors. 4. Discussion
3.4. PRMT1 ablation ameliorates inflammation and oxidative stress in LPS-exposed astrocytes via Nrf-2 Astrocytes were isolated from PRMT1þ/þ and PRMT1/ mice, and the expression results were verified using western blot analysis (Fig. 4A). LPS treatment significantly stimulated the expression of PRMT1 from mRNA and protein levels (Fig. 4B and C). GFAP expression levels induced by LPS were obviously prevented by PRMT1 knockout using immunofluorescent analysis (Fig. 4D). The mRNA levels of IL-1b and TNF-a triggered by LPS exposure were significantly down-regulated by PRMT1 deletion in AST, which was accompanied with a remarkable decrease in phosphoeNFekB expression (Fig. 4E and F). DCF-DA analysis demonstrated that PRMT1/ markedly reduced ROS production in LPS-incubated AST (Fig. 4G). Inversely, Nrf-2 expression levels inhibited by LPS were markedly rescued by PRMT1 deficiency (Fig. 4H). Further, effects of
Accumulating evidence suggests that inflammation, increased cytokine levels and oxidative stress are related to anxiety- and depression-like symptoms and neuropsychological disturbances in humans [3e5]. In rodent animals, increasing studies have proved that depressive-like behaviors could be induced by cytokines or cytokine inducers, including LPS administration or chronic mild stress [6]. As reported, targeting the neuroinflammatory disturbances has been acknowledged as a potential avenue for prevention of depression [24]. Previous study of healthy men indicated that immune activation by LPS administration produces changes in emotional states and an elevation of reported depressive symptoms [25,26]. Furthermore, acute activation of the peripheral or central innate immune system in animals studies, via the LPS administration, results in a strong immune response and depressive-like behavior, as determined by elevated immobility in the tail
Fig. 3. Knockout of PRMT1 attenuates LPS-induced inflammation and oxidative stress in hippocampus of mice. (A&B) ELISA analysis of IL-1b and TNF-a in hippocampus and PFC of mice. (C&D) RT-qPCR determination of IL-1b and TNF-a in hippocampus and PFC of mice. (E) Western blot measurement of peNFekB in hippocampus of mice. (F) Immunofluorescent analysis of peNFekB in hippocampus sections. Determination of (G) MDA levels, (H) SOD and (I) GSH-pX activities in hippocampus of mice. (J) Immunofluorescent analysis of Nrf-2 in hippocampus tissues. Data were expressed as the mean ± S.E.M (n ¼ 8 per group). #P < 0.05, ##P < 0.01 and ###P < 0.001 versus Con/PRMTþ/þ group; þ P < 0.05, þþP < 0.01 and þþþP < 0.001 versus LPS/PRMTþ/þ group.
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
435
Fig. 4. PRMT1 ablation ameliorates inflammation and oxidative stress in LPS-exposed astrocytes via Nrf-2. (A) Astrocytes (AST) were isolated from PRMT1þ/þ and PRMT1/ mice, followed by western blot analysis of cellular PRMT1. (B&C) AST cells isolated from PRMT1þ/þ mice were treated with 100 ng/ml LPS for 24 h, followed by RT-qPCR and western blot analysis of PRMT1. ###P < 0.001 versus Con group. (D) Immunofluorescent analysis of GFAP in 24 h of LPS (100 ng/ml)-incubated AST isolated from PRMT1þ/þ and PRMT1/ mice. (E) RT-qPCR analysis of IL-1b and TNF-a in AST treated with 100 ng/ml LPS for 24 h. (F) Western blot measurement of peNFekB in AST stimulated by 24 h of LPS (100 ng/ml). (G) DCF-DA analysis in AST treated as indicated. (H) Western blot analysis of Nrf-2 in LPS-incubated AST. ##P < 0.01 and ###P < 0.001 versus Con/PRMTþ/þ group; þþP < 0.01 and þþþ P < 0.001 versus LPS/PRMTþ/þ group. (I) AST cells were treated with the indicated concentrations of ML385 for 4 h, followed by Nrf-2 calculation using western blotting analysis. AST cells were pre-treated with 10 mM of ML385 (Nrf-2 suppressor) for 4 h, and then were subjected to LPS (100 ng/ml) treatment for another 24 h. Further studies as followings were carried out. (J) RT-qPCR analysis of IL-1b and TNF-a in AST cells. (K) Western blot analysis of peNFekB. (L) DCF-DA analysis of AST cells. ##P < 0.01 versus LPS/PRMTþ/þ group; þ P < 0.05 and þþP < 0.01 versus LPS/PRMT/- group. Data were expressed as the mean ± S.E.M (n ¼ 6 per group in vitro).
suspension test and forced swim test, a repression of consumption of a sweetened solution [27e29]. In addition, changes in dendritic length and spine density in PFC and hippocampus are participated in the pathophysiology of depression [30]. In the study, we also found that LPS markedly induced anxiety- and depression-like symptoms in mice through various tests, including locomotion test, tail-suspension test, forced swim test, sucrose preference test, and open field test. However, LPS-induced depression behaviors were effectively alleviated in LPS-treated mice in the absence of PRMT1. What's more, alterations in dendritic length and spine density in PFC and hippocampus of LPS mice were improved by PRMT1 knockout. Thus, PRMT1 was involved in anxiety- and
depression-like behaviors induced by LPS in mice. Mechanistically, PRMT1 knockout markedly suppressed LPS-triggered inflammation and oxidative stress in hippocampus of mice via promoting peNFekB and repressing Nrf-2 expressions, respectively. Of note, the in vitro study suggested that PRMT1 knockout-reduced inflammation and ROS generation were evidently restored by the pre-treatment of ML385, a Nrf-2 inhibitor, in LPS-incubated AST. Therefore, restraining PRMT1 expression showed an antidepressant-like effect. PRMTs have crucial regulatory functions as transcription factor co-modulators [31]. As a member of PRMTs, PRMT1 has been involved in cytokines-induced lung epithelial cells and fibroblasts,
436
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
and is associated with the progression of both inflammation and remodeling in asthma [10,32]. Depression is along with the activation of the inflammatory-response, and elevated release of proinflammatory cytokines, such as IL-6, IL-1b and TNF-a, may play a critical role in the pathophysiology of depressive disorders [33]. Promoted generation of inflammatory cytokines enhances the activation of glia cells, including astrocytes and microglia cells, which could, in turn, accelerate neuroinflammatory damage [34]. Our results suggested that PRMT1 deficiency significantly decreased LPS-induced GFAP and Iba-1 expression, demonstrating the inactivation of glia cells [35]. Consistently, the release or expression of IL-1b and TNF-a in hippocampus of LPS-challenged mice was evidently impeded by PRMT1 deletion, which was accompanied with down-regulated expression of peNFekB. Thus, consistent with previous study, PRMT1 played an essential role in regulating inflammatory response, thereby influencing LPS-elicited anxiety- and depression-like behaviors. Additionally, PRMT1 is involved in ROS-and RAS-regulated diabetic retinopathy [36]. PRMT1-associated oxidative stress was involved in the cardiovascular risk [37]. Moreover, a growing body of data has indicated that ROS also play a critical role in the pathogenesis of neuropsychiatric disorders [38]. Previous reports also demonstrated that oxidative stress was activated during neuroinflammation [39]. The observation was confirmed by our findings, which indicated that several markers reflecting the status of oxidative stress, including MDA, SOD and GSH-pX, were evidently changed by LPS administration, suggesting the presence of a higher oxidative response and a lower antioxidant status. Notably, PRMT1 knockout significantly alleviated LPS-induced oxidative stress via the expression of Nrf-2. Excessive studies have reported that Nrf-2 is involved in oxidative and inflammatory damage [21,40]. Here, we also found that suppressing Nrf-2 expression abolished PRMT1 knockout-attenuated inflammation and ROS generation in LPSstimulated AST. Therefore, PRMT1 could meditate Nrf-2 expression to influence LPS-induced inflammation and oxidative stress, which subsequently affected anxiety- and depression-like behaviors. In conclusion, our data illustrated that LPS exposure in mice resulted in neuroinflammatory and oxidative responses, and importantly the process could be restrained by the knockout of PRMT1 at least partly dependent on Nrf-2 signaling. Thereby, PRMT1 could be served as an important therapeutic target for developing novel and effective treatment to ameliorate depression in patients. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.032. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.032. References [1] E. Pettersson, et al., Common psychiatric disorders share the same genetic origin: a multivariate sibling study of the Swedish population, Mol. Psychiatry 21 (5) (2016) 717. [2] Y. Wang, et al., Influence of anti-depression therapy on inflammatory cytokines and quality of life in patients with acute coronary syndrome complicating depression, Int. J. Lab. Med. 38 (2017) 1365e1367. [3] A.L. Lopresti, et al., A review of peripheral biomarkers in major depression: the potential of inflammatory and oxidative stress biomarkers, Prog. Neuro Psychopharmacol. Biol. Psychiatry 48 (2014) 102e111. [4] Y. Dowlati, et al., A meta-analysis of cytokines in major depression, Biol.
Psychiatry 67 (2010) 446e457. [5] S.A. Hiles, et al., A meta-analysis of differences in IL-6 and IL-10 between people with and without depression: exploring the causes of heterogeneity, Brain Behav. Immun. 26 (2012) 1180e1188. [6] B.S.F. Mello, et al., Effects of doxycycline on depressive-like behavior in mice after lipopolysaccharide (LPS) administration, J. Psychiatr. Res. 47 (2013) 1521e1529. re, et al., Methyltransferase PRMT1 Is a [7] S. Benhenda, A. Ducroux, L. Rivie binding partner of HBx and a negative regulator of hepatitis B virus transcription, J. Virol. 87 (8) (2013) 4360e4371. [8] X. Zhao, et al., Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity, Genes Dev. 22 (2008) 640e653. [9] M. Le Romancer, et al., Regulation of estrogen rapid signaling through arginine methylation by PRMT1, Mol. Cell 31 (2008) 212e221. [10] Q. Sun, et al., PRMT1 upregulated by epithelial proinflammatory cytokines participates in COX2 expression in fibroblasts and chronic antigen-induced pulmonary inflammation, J. Immunol. (2015), 1402465. [11] M.L. Onozato, et al., Expression of DDAH and PRMT isoforms in the diabetic rat kidney; effects of angiotensin II receptor blocker, Diabetes 57 (2007). [12] W. Shao, et al., Suppression of neuroinflammation by astrocytic dopamine D2 receptors via aB-crystallin, Nature 494 (2013) 90. [13] Q. Ren, et al., Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress, Proc. Natl. Acad. Sci. 113 (2016) E1944eE1952. [14] Q. Ren, et al., BDNFeTrkB signaling in the nucleus accumbens shell of mice has key role in methamphetamine withdrawal symptoms, Transl. Psychiatry 5 (2015), e666. [15] C. Yang, et al., R-ketamine: a rapid-onset and sustained antidepressant without psychotomimetic side effects, Transl. Psychiatry 5 (2015), e632. [16] M. Li, et al., Lentivirus-mediated interleukin-1b (IL-1b) knock-down in the hippocampus alleviates lipopolysaccharide (LPS)-induced memory deficits and anxiety-and depression-like behaviors in mice, J. Neuroinflammation 14 (2017) 190. [17] M.P. Kaster, et al., Depressive-like behavior induced by tumor necrosis factora in mice, Neuropharmacology 62 (2012) 419e426. [18] J. Zhang, et al., Antidepressant effects of TrkB ligands on depression-like behavior and dendritic changes in mice after inflammation, Int. J. Neuropsychopharmacol. 18 (2015). [19] S.A. Villeda, et al., Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice, Nat. Med. 20 (2014) 659. [20] T.V. Lipina, et al., Disrupted-in-schizophrenia-1 Gln31Leu polymorphism results in social anhedonia associated with monoaminergic imbalance and reduction of CREB and b-arrestin-1, 2 in the nucleus accumbens in a mouse model of depression, Neuropsychopharmacology 38 (2013) 423. [21] J.P. Guilloux, et al., Molecular evidence for BDNF-and GABA-related dysfunctions in the amygdala of female subjects with major depression, Mol. Psychiatry 17 (2012) 1130. [22] M.V. Doucet, et al., Small-molecule inhibitors at the PSD-95/nNOS interface have antidepressant-like properties in mice, Neuropsychopharmacology 38 (2013) 1575. [23] S.H. Seo, et al., Fisetin inhibits TNF-a-induced inflammatory action and hydrogen peroxide-induced oxidative damage in human keratinocyte HaCaT cells through PI3K/AKT/Nrf-2-mediated heme oxygenase-1 expression, Int. Immunopharmacol. 29 (2015) 246e253. [24] M. Ma, et al., Antidepressant effects of combination of brexpiprazole and fluoxetine on depression-like behavior and dendritic changes in mice after inflammation, Psychopharmacology 234 (2017) 525e533. [25] S.M. Clark, et al., Dissociation between sickness behavior and emotionality during lipopolysaccharide challenge in lymphocyte deficient Rag2/ mice, Behav. Brain Res. 278 (2015) 74e82. [26] L. Ge, et al., Resveratrol abrogates lipopolysaccharide-induced depressive-like behavior, neuroinflammatory response, and CREB/BDNF signaling in mice, Eur. J. Pharmacol. 768 (2015) 49e57. [27] Y. Ohgi, et al., Effects of antidepressants on alternations in serum cytokines and depressive-like behavior in mice after lipopolysaccharide administration, Pharmacol. Biochem. Behav. 103 (2013) 853e859. [28] Z. Wang, et al., The effects of curcumin on depressive-like behavior in mice after lipopolysaccharide administration, Behav. Brain Res. 274 (2014) 282e290. [29] A.K. Walker, et al., NMDA receptor blockade by ketamine abrogates lipopolysaccharide-induced depressive-like behavior in C57BL/6J mice, Neuropsychopharmacology 38 (2013) 1609. [30] X. Deng, et al., Lipolysaccharide-induced neuroinflammation is associated with Alzheimer-like amyloidogenic axonal pathology and dendritic degeneration in rats, Adv. Alzheimer's Dis. 3 (2014) 78. [31] B. Cha, et al., Protein arginine methyltransferases (PRMTs) as therapeutic targets, Expert Opin. Ther. Targets 16 (2012) 651e664. [32] Q. Sun, et al., Upregulated protein arginine methyltransferase 1 by IL-4 increases eotaxin-1 expression in airway epithelial cells and participates in antigen-induced pulmonary inflammation in rats, J. Immunol. (2012), 1102635. [33] D.T. Skelly, et al., A systematic analysis of the peripheral and CNS effects of systemic LPS, IL-1b, TNF-a and IL-6 challenges in C57BL/6 mice, PLoS One 8 (2013), e69123. [34] Y.Y. Zhang, et al., Atorvastatin attenuates the production of IL-1b, IL-6, and
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437 TNF-a in the hippocampus of an amyloid b1-42-induced rat model of Alzheimer's disease, Clin. Interv. Aging 8 (2013) 103. [35] D.M. Norden, et al., Sequential activation of microglia and astrocyte cytokine expression precedes increased iba-1 or GFAP immunoreactivity following systemic immune challenge, Glia 64 (2016) 300e316. [36] D.I. Kim, et al., PRMT1 and PRMT4 regulate oxidative stress-induced retinal pigment epithelial cell damage in SIRT1-dependent and SIRT1-independent manners, Oxidative med. cell. longev. (2015) 2015. [37] C. Savoia, et al., Countervailing vascular effects of rosiglitazone in high cardiovascular risk mice: role of oxidative stress and PRMT-1, Clin. Sci. 118
437
(2010) 583e592. [38] D. Ben-Shachar, et al., Dopamine toxicity involves mitochondrial complex I inhibition: implications to dopamine-related neuropsychiatric disorders, Biochem. Pharmacol. 67 (2004) 1965e1974. [39] N. Bakunina, et al., Immune mechanisms linked to depression via oxidative stress and neuroprogression, Immunology 144 (2015) 365e373. [40] C.H. Kang, et al., 5-Hydroxy-3, 6, 7, 8, 30 40 -hexamethoxyflavone inhibits nitric oxide production in lipopolysaccharide-stimulated BV2 microglia via NF-kB suppression and Nrf-2-dependent heme oxygenase-1 induction, Food Chem. Toxicol. 57 (2013) 119e125.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Protein arginine methyltransferase-1 deficiency restrains depressionlike behavior of mice by inhibiting inflammation and oxidative stress via Nrf-2 Hongbo Liu, Jinghui Jiang, Lili Zhao* Department of Paediatrics, Liaocheng People's Hospital, Liaocheng, 252000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 July 2019 Accepted 7 August 2019 Available online 3 September 2019
Current evidence indicates that depression is accompanied by the activation of inflammatory response and oxidative stress. Protein arginine methyltransferase 1 (PRMT1) is a histone methyltransferase that methylates Arg3 on histone H4, playing crucial role in regulating various pathological processes. In the study, we attempted to explore the effects of PRMT1 on animal model with depression through a single administration of lipopolysaccharide (LPS). Our results indicated that PRMT1 knockout (PRMT1/) improved LPS-induced anxiety- and depressive-like behavior, along with up-regulated expression levels of brain-derived neurotrophic factor (BDNF) and PSD-95. Furthermore, PRMT1 deficiency significantly improved LPS-induced changes in dendritic spine density in the areas of prefrontal cortex (PFC), CA3 and dentate gyrus (DG), and nucleus accumbens (NAc). In addition, PRMT1 deletion ameliorated the neuroinflammatory responses, as evidenced by the reduced expression of interleukin 1b (IL-1b) and tumor necrosis factor (TNF)-a, which might be through repressing nuclear factor-kB (NF-kB) signaling. Moreover, oxidative stress induced by LPS was alleviated by PRMT1 knockout in hippocampus of mice at least partly via promoting Nrf-2 expressions. The anti-depressant effects of PRMT1 inhibition were verified in LPS-incubated astrocytes. Importantly, we found that PRMT1 knockout-alleviated inflammation and oxidative stress triggered by LPS were significantly recovered by the suppression of Nrf-2. Therefore, Nrf2 was markedly involved in PRMT1-regulated depression-like behavior. Taken together, the results indicated that PRMT1 might be an important therapeutic target for developing effective treatment to prevent depressive-like behavior. © 2019 Published by Elsevier Inc.
Keywords: Depression PRMT1 Inflammation Oxidative stress Nrf-2
1. Introduction Depression is one of the most common psychiatric disorders worldwide [1]. Despite the precise molecular mechanism underlying the pathophysiology of depression is unclear, multiple lines of evidence illustrate that inflammatory response and oxidative stress play a critical role in the pathophysiology of depression [2,3]. Also, a meta-analysis indicates higher blood levels of pro-inflammatory cytokines in drug-free depressed patients when compared to healthy controls [4,5]. Presently, peripheral administration of the bacterial endotoxin, LPS, triggers depression-like behavior in mice after the induction of inflammation, as well as oxidative stress [6]. Therefore, finding significant target associated with inflammation and oxidative stress might be effective for developing therapeutic strategy against depressive symptoms.
* Corresponding author. E-mail address: [email protected] (L. Zhao). https://doi.org/10.1016/j.bbrc.2019.08.032 0006-291X/© 2019 Published by Elsevier Inc.
Protein arginine methyltransferase-1 (PRMT1), methylating both histones and key cellular proteins, has been served as a crucial modulator of various cellular processes, such as signal transduction, protein and protein interaction, as well as transcriptional modulation [7,8]. For instance, PRMT1 played an essential role in gene activation and metamorphosis triggered by ligand activated thyroid hormone receptor [9]. In addition, PRMT1 expression participates in fibroblast proliferation and chronic airway inflammation in Aginduced pulmonary inflammation through the regulation of remodeling associated cytokines [10]. In vitro studies also suggested that angiotensin-II markedly induced PRMT1 expression and endothelial cell activation, thus producing reactive oxygen species (ROS) [11]. Thus, PRMT1 is involved in the meditation of inflammatory response and ROS generation. However, its influences on anxiety- and depression-like behaviors have not been investigated and reported. The purpose of the study is to explore if PRMT1 could affect LPSinduced animal model with depression. The results suggested that
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
PRMT1 deletion significantly improved anxiety- and depressionlike behaviors induced by LPS in mice. Inflammatory response and oxidative stress induced by LPS were obviously ameliorated by the knockout of PRMT1 in hippocampus of mice. Notably, the in vitro experiments using LPS-treated astrocytes demonstrated that PRMT1-meditated inflammatory and oxidative damage was largely dependent on Nrf-2. Together, our study elucidated that PRMT1 could be served as a therapeutic candidate to develop effective treatment for alleviating depression. 2. Materials and methods 2.1. Animals and treatments Wild type C57BL/6 mice (adult male, PRMT1þ/þ) weighing 22e25 g were obtained from the Vital River Laboratories (Beijing, China). PRMT1 knockout (PRMT1/) mice with C57BL/6 background were constructed and purchased from Model Animal Research Center of Nanjing University (Nanjing, China). All mice were housed in a 12-h dark/light cycle, temperature (20 ± 2 C) and humidity controlled environment with free access to water and food. 0.5 mg/kg of LPS (L-4130, serotype 0111:B4, Sigma-Aldrich, USA) was dissolved in distilled water. Saline (10 ml/kg) or LPS (0.5 mg/kg) was administered intraperitoneally (i.p.) to mice. Then behavior was measured following saline or LPS administration for 24 h. The experimental procedure was approved by the Animal Care and Use Committee of the First Affiliated Hospital of Harbin Medical University (Harbin, China). All efforts were made to reduce the number of animal used and their suffering. 2.2. Cells and incubation Astrocytes (AST) from PRMT1/ and PRMT1þ/þ mice were prepared essentially as described in the previous reports [12]. AST was then collected and seeded in DMEM/F12 medium (GIBCO Corporation, Gaithersburg, MD, USA), supplemented with 10% FBS (GIBCO Corporation), 1 105 U/L streptomycin sulfate with a concentration 1 106/mL at 37 C with 5% CO2. LPS and/or ML385 MedChem Express (Monmouth Junction, NJ, USA) were exposed to AST for further study.
431
homogenized in ice-cold RIPA buffer with Complete Protease Inhibitor (Roche, USA). The dissolved proteins were harvested after centrifugation at 12,000 g for 15 min at 4 C. Then, the supernatant was collected. A quantity of 25e50 mg total proteins was loaded onto a 10e12% SDS-PAGE, electrophoretically transferred to polyvinylidene difluoride membrane (PVDF) and probed with the following primary antibodies: phosphoeNFekB p65 (1:1000, Cell Signaling), NF-kB (1:1000, Cell Signaling). Nrf-2 (1:1000, Abcam, USA), GFAP (1:1000, Abcam), PRMT1 (1:1000, Abcam), and Iba-1 (1:1000, Santa Cruz Biotechnology, Inc.). GAPDH (1:1000, Abcam) was used as an internal control. Secondary antibodies were horseradish peroxidase (HRP) conjugated to goat/mouse antirabbit IgG (1:6000, Sigma Aldrich). The membranes were developed using an enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology). 2.6. Biochemical measurements Concentrations of MDA (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), SOD (Nanjing Jiancheng Bioengineering Institute), GSH-pX (Nanjing Jiancheng Bioengineering Institute), TNF-a (R&D System, USA) and IL-1b (R&D System) were quantified using commercial kits according to the manufacturer's protocol. 2.7. Immunofluorescent analysis The brain and AST cells were fixed with 4% paraformaldehyde. Then, the brain tissue was treated with 30% sucrose at 4 C for 48 h. Brain tissue was sectioned with a thickness of 10 mm and pasted into microscope slides, followed by blocking with buffer (BSA, PBS with 0.3% Triton X-100) for 1 h. The sections were incubated with anti-NeuN (1:200, Abcam), GFAP (1:200, Abcam), phosphoeNFekB p65 (1:200, Cell Signaling) or Nrf-2 (Abcam) overnight at 4 C. The sections were rinsed with PBS and incubated with fluorescent secondary antibodies (1:500, Invitrogen), for 1 h at room temperature. Nuclei were stained with DAPI (Solarbio, Beijing, China) for 15 min, mounted onto slides and coverslips with ProLong mounting medium (Invitrogen). The representative images were captured using a fluorescent microscope. 2.8. DCF-DA analysis
2.3. Behavior analysis Behavioral tests, including locomotion, tail suspension test (TST), forced swimming test, sucrose preference test and open field test, were performed as previously reported [13e17]. All behavioral tests were conducted during the illuminated part of the cycle (between 9:00 and 12:00 a.m.). Behavior experiments were monitored by a trained observer blind to treatment. 2.4. Real time-quantitative PCR (RT-qPCR) analysis Total RNA was extracted from the hippocampus and cells using Trizol reagent (Gibco) according to the manufacturer's instructions. Identical amounts of RNA were reversely transcribed into cDNA with a commercial RT-PCR kit (Fermentas, Vilnius, Lithuania) according to the manufacturer's instructions. Real-time RT-PCR was carried out with 1 ml cDNA on real-time system (Analtik Jena. AG. Germany) using an UltraSYBR mixture (CoWin Bioscience Co., Beijing, China) and normalized to b-actin. Protocol of the real-time PCR was performed. The primers were listed in Supplementary table 1. 2.5. Western blot analysis Frozen tissue from the entire hippocampus and cells was
ROS levels in AST were determined using the specific probe DCFDA (Life Technologies, Invitrogen, USA) following the manufacturer's instructions. The stained AST cells were analyzed. 2.9. Immunohistochemical analysis Brain tissues were cut with 5 mm thick coronal sections on a microtome, then de-waxed with xylene and rehydrated with ethanol. After washing with PBS, all sections were stained with 0.1% Cresyl violet (Sigma Aldrich, USA) for 30 min at 37 C. The representative images were captured using a light microscope for surviving nerve cells in hippocampus area. 2.10. Golgi staining Golgi staining was carried out using the FD Rapid GolgiStain™ Kit (FD Neuro Technologies, Inc., Columbia, USA), following the manufacturer's instructions as previously described [14,18,19]. Representative images of dendrites within CA1, CA3, and DG of the hippocampus, prelimbic and inflalimbic areas of medial prefrontal cortex (mPFC), and shell and core of nucleus accumbens (NAc) were captured with a Keyence BZ-9000 GenerationIImicroscope (Osaka, Japan). Spines were analyzed along CA1, CA3, DG, prelimbic and
432
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
inflalimbic areas of mPFC, as well as shell and core of NAc dendrites as previously indicated [18,20]. 2.11. Statistics Data were presented as the mean ± S.E.M. Statistical analysis of data was carried out by one-way analysis of variance (ANOVA), followed by post-hoc Tukey test. Differences were considered statistically significant if the p value was P < 0.05. 3. Results 3.1. The influence of PRMT1 on anxiety- and depression-like behaviors induced by LPS in mice In the regard, the effects of PRMT1 on anxiety- and depressionlike behaviors induced by LPS were investigated. In the locomotion
test, no significant differences were observed (Fig. 1A). TST and FST results suggested that PRMT1 knockout significantly attenuated the increased immobility time in mice after LPS treatment (Fig. 1B and C). Moreover, PRMT1 deletion produced a complementary upregulation in swimming time compared to LPS/PRMT1þ/þ group (Fig. 1D). In the sucrose preference test, LPS-challenged PRMT1þ/þ mice showed a marked decrease in sucrose preference index as compared to saline-treated mice, whereas PRMT1 knockout significantly improved the sucrose preference index in LPS-treated mice (Fig. 1E). Open field test indicated that the decrease in line crossings produced by LPS was markedly prevented by PRMT1 ablation (Fig. 1F). Moreover, the reduction in recognition index induced by LPS was significantly improved by PRMT1 knockout (Fig. 1G). As shown in Fig. 1H and I, the duration in the open arms and entries into the open arms reduced by LPS administration were improved in PRMT1/ mice. BDNF and PSD-95 are the prominent regulators of neuronal survival, growth, and differentiation during
Fig. 1. The influence of PRMT1 on anxiety- and depression-like behaviors induced by LPS in mice. (A) Locomotion (LST), (B) tail-suspension test (TST), (C) and forced swim test (FST) were performed 2 h, 4 h, and 6 h after 24 h of LPS administration. (D) Swimming time in each group of mice was measured. (E) Percentage of sucrose consumption in the sucrose preference test (SPT). (F) The number of crossing in open field test. (G) Recognition index for novel objects induced by LPS in mice. (H) Duration in the open arms. (I) The entries into the open arms. RT-qPCR analysis of (J) BDNF and (K) PSD-95 in brain areas of PFC, NAc, CA1, CA3 and DG in mice. Data were expressed as the mean ± S.E.M (n ¼ 15 per group for behavior tests and n ¼ 8 for RT-qPCR analysis). #P < 0.05 and ##P < 0.01 versus Con/PRMTþ/þ group; þP < 0.05 and þþP < 0.01 versus LPS/PRMTþ/þ group.
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
development [21,22]. As described in Fig. 1J and K, the expression levels of BDNF and PSD-95 were markedly decreased by LPS challenge in brain areas of PFC, NAc, CA3 and DG, which were reversed by the loss of PRMT1. The levels of BDNF and PSD-95 were not affected by LPS or PRMT1 deletion in CA1 area. The results above demonstrated that PRMT1 was involved in anxiety- and depression-like behaviors induced by LPS in mice.
433
dendritic spine density in shell and core of NAc in PRMT1þ/þ mice; however, the changes induced by LPS were reversed by PRMT1 deletion (Fig. 2A and B). The number of surviving cells was obviously reduced in hippocampus of LPS/PRMT1þ/þ mice, while being markedly alleviated in mice lacking of PRMT1 expression (Fig. 2C and D). RT-qPCR and western blot results indicated that LPSpromoted expression levels of GFAP and Iba-1 were clearly downregulated in PRMT1/ mice (Fig. 2E and F).
3.2. PRMT1 deletion alleviates brain injury of LPS-induced mice Golgi staining data suggested that LPS significantly reduced dendritic spine density in the prelimbic and infralimbic regions of PFC, CA3 and DG, but not in the CA1, of hippocampus, and enhanced
3.3. Knockout of PRMT1 attenuates LPS-induced inflammation and oxidative stress in hippocampus of mice The levels of IL-1b and TNF-a were significantly increased after
Fig. 2. PRMT1 deletion alleviates brain injury of LPS-induced mice. (A&B) Golgi staining in the brain regions, including prelimbic and infralimbic regions of PFC, NAc core and shell, CA1, CA3 and DG of hippocampus) was performed. Representative images and data of Golgi staining in the brain regions of mice. (C) Cresyl violet staining of CA1 area in hippocampus of mice. (D) Immunofluorescent analysis of NeuN in CA1 area of hippocampus. (E&F) RT-qPCR and western blot analysis of GFAP and Iba-1 in hippocampus of mice. Data were expressed as the mean ± S.E.M (n ¼ 8 per group). #P < 0.05, ##P < 0.01 and ###P < 0.001 versus Con/PRMTþ/þ group; þP < 0.05 and þþP < 0.01 versus LPS/PRMTþ/þ group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
434
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
24 h of LPS administration both in the hippocampus and PFC compared with those in control group of PRMT1þ/þ mice. Meanwhile, PRMT1 deficiency evidently decreased IL-1b and TNF-ain the hippocampus and PFC in LPS-challenged mice using RT-qPCR and ELISA analysis (Fig. 3AeD). In mice treated with LPS, phosphoeNFekB expressions were markedly increased in the hippocampus, whereas PRMT1 knockout showed significant inhibitory effect on phosphoeNFekB expression (Fig. 3E and F). The significant increase of MDA in hippocampus induced by LPS was markedly prevented by the loss of PRMT1 (Fig. 3G). In contrast, LPSsuppressed SOD and GSH-pX activities in hippocampus of mice were significantly restored by PRMT1 deletion (Fig. 3H and I). Immunofluorescent analysis suggested that Nrf-2 expression levels were markedly reduced by LPS challenge in hippocampus of mice, whereas being improved in PRMT1/ mice (Fig. 3J).
Nrf-2 on PRMT1-regulated central injury were explored considering the essential role of Nrf-2 in the meditation of inflammatory response and oxidative stress [23]. Next, Nrf-2 expression in AST was repressed by adding ML385, a significant Nrf-2 inhibitor (Fig. 4I). Importantly, PRMT1 knockout-reduced expression levels of IL-1b and TNF-a in LPS-exposed AST were regained by ML385 pre-treatment (Fig. 4J). Also, ML385 pre-treatment markedly recovered the expression of phosphoeNFekB in LPS-treated AST in the absence of PRMT1 (Fig. 4K). Furthermore, PRMT1/-suppressed ROS generation in LPS-exposed cells was markedly restored by ML385 pre-treatment (Fig. 4L). The results above indicated that PRMT1-regulated inflammation and oxidative stress in LPS-treated astrocytes were partly dependent on Nrf-2, thereby modulating anxiety- and depression-like behaviors. 4. Discussion
3.4. PRMT1 ablation ameliorates inflammation and oxidative stress in LPS-exposed astrocytes via Nrf-2 Astrocytes were isolated from PRMT1þ/þ and PRMT1/ mice, and the expression results were verified using western blot analysis (Fig. 4A). LPS treatment significantly stimulated the expression of PRMT1 from mRNA and protein levels (Fig. 4B and C). GFAP expression levels induced by LPS were obviously prevented by PRMT1 knockout using immunofluorescent analysis (Fig. 4D). The mRNA levels of IL-1b and TNF-a triggered by LPS exposure were significantly down-regulated by PRMT1 deletion in AST, which was accompanied with a remarkable decrease in phosphoeNFekB expression (Fig. 4E and F). DCF-DA analysis demonstrated that PRMT1/ markedly reduced ROS production in LPS-incubated AST (Fig. 4G). Inversely, Nrf-2 expression levels inhibited by LPS were markedly rescued by PRMT1 deficiency (Fig. 4H). Further, effects of
Accumulating evidence suggests that inflammation, increased cytokine levels and oxidative stress are related to anxiety- and depression-like symptoms and neuropsychological disturbances in humans [3e5]. In rodent animals, increasing studies have proved that depressive-like behaviors could be induced by cytokines or cytokine inducers, including LPS administration or chronic mild stress [6]. As reported, targeting the neuroinflammatory disturbances has been acknowledged as a potential avenue for prevention of depression [24]. Previous study of healthy men indicated that immune activation by LPS administration produces changes in emotional states and an elevation of reported depressive symptoms [25,26]. Furthermore, acute activation of the peripheral or central innate immune system in animals studies, via the LPS administration, results in a strong immune response and depressive-like behavior, as determined by elevated immobility in the tail
Fig. 3. Knockout of PRMT1 attenuates LPS-induced inflammation and oxidative stress in hippocampus of mice. (A&B) ELISA analysis of IL-1b and TNF-a in hippocampus and PFC of mice. (C&D) RT-qPCR determination of IL-1b and TNF-a in hippocampus and PFC of mice. (E) Western blot measurement of peNFekB in hippocampus of mice. (F) Immunofluorescent analysis of peNFekB in hippocampus sections. Determination of (G) MDA levels, (H) SOD and (I) GSH-pX activities in hippocampus of mice. (J) Immunofluorescent analysis of Nrf-2 in hippocampus tissues. Data were expressed as the mean ± S.E.M (n ¼ 8 per group). #P < 0.05, ##P < 0.01 and ###P < 0.001 versus Con/PRMTþ/þ group; þ P < 0.05, þþP < 0.01 and þþþP < 0.001 versus LPS/PRMTþ/þ group.
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
435
Fig. 4. PRMT1 ablation ameliorates inflammation and oxidative stress in LPS-exposed astrocytes via Nrf-2. (A) Astrocytes (AST) were isolated from PRMT1þ/þ and PRMT1/ mice, followed by western blot analysis of cellular PRMT1. (B&C) AST cells isolated from PRMT1þ/þ mice were treated with 100 ng/ml LPS for 24 h, followed by RT-qPCR and western blot analysis of PRMT1. ###P < 0.001 versus Con group. (D) Immunofluorescent analysis of GFAP in 24 h of LPS (100 ng/ml)-incubated AST isolated from PRMT1þ/þ and PRMT1/ mice. (E) RT-qPCR analysis of IL-1b and TNF-a in AST treated with 100 ng/ml LPS for 24 h. (F) Western blot measurement of peNFekB in AST stimulated by 24 h of LPS (100 ng/ml). (G) DCF-DA analysis in AST treated as indicated. (H) Western blot analysis of Nrf-2 in LPS-incubated AST. ##P < 0.01 and ###P < 0.001 versus Con/PRMTþ/þ group; þþP < 0.01 and þþþ P < 0.001 versus LPS/PRMTþ/þ group. (I) AST cells were treated with the indicated concentrations of ML385 for 4 h, followed by Nrf-2 calculation using western blotting analysis. AST cells were pre-treated with 10 mM of ML385 (Nrf-2 suppressor) for 4 h, and then were subjected to LPS (100 ng/ml) treatment for another 24 h. Further studies as followings were carried out. (J) RT-qPCR analysis of IL-1b and TNF-a in AST cells. (K) Western blot analysis of peNFekB. (L) DCF-DA analysis of AST cells. ##P < 0.01 versus LPS/PRMTþ/þ group; þ P < 0.05 and þþP < 0.01 versus LPS/PRMT/- group. Data were expressed as the mean ± S.E.M (n ¼ 6 per group in vitro).
suspension test and forced swim test, a repression of consumption of a sweetened solution [27e29]. In addition, changes in dendritic length and spine density in PFC and hippocampus are participated in the pathophysiology of depression [30]. In the study, we also found that LPS markedly induced anxiety- and depression-like symptoms in mice through various tests, including locomotion test, tail-suspension test, forced swim test, sucrose preference test, and open field test. However, LPS-induced depression behaviors were effectively alleviated in LPS-treated mice in the absence of PRMT1. What's more, alterations in dendritic length and spine density in PFC and hippocampus of LPS mice were improved by PRMT1 knockout. Thus, PRMT1 was involved in anxiety- and
depression-like behaviors induced by LPS in mice. Mechanistically, PRMT1 knockout markedly suppressed LPS-triggered inflammation and oxidative stress in hippocampus of mice via promoting peNFekB and repressing Nrf-2 expressions, respectively. Of note, the in vitro study suggested that PRMT1 knockout-reduced inflammation and ROS generation were evidently restored by the pre-treatment of ML385, a Nrf-2 inhibitor, in LPS-incubated AST. Therefore, restraining PRMT1 expression showed an antidepressant-like effect. PRMTs have crucial regulatory functions as transcription factor co-modulators [31]. As a member of PRMTs, PRMT1 has been involved in cytokines-induced lung epithelial cells and fibroblasts,
436
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437
and is associated with the progression of both inflammation and remodeling in asthma [10,32]. Depression is along with the activation of the inflammatory-response, and elevated release of proinflammatory cytokines, such as IL-6, IL-1b and TNF-a, may play a critical role in the pathophysiology of depressive disorders [33]. Promoted generation of inflammatory cytokines enhances the activation of glia cells, including astrocytes and microglia cells, which could, in turn, accelerate neuroinflammatory damage [34]. Our results suggested that PRMT1 deficiency significantly decreased LPS-induced GFAP and Iba-1 expression, demonstrating the inactivation of glia cells [35]. Consistently, the release or expression of IL-1b and TNF-a in hippocampus of LPS-challenged mice was evidently impeded by PRMT1 deletion, which was accompanied with down-regulated expression of peNFekB. Thus, consistent with previous study, PRMT1 played an essential role in regulating inflammatory response, thereby influencing LPS-elicited anxiety- and depression-like behaviors. Additionally, PRMT1 is involved in ROS-and RAS-regulated diabetic retinopathy [36]. PRMT1-associated oxidative stress was involved in the cardiovascular risk [37]. Moreover, a growing body of data has indicated that ROS also play a critical role in the pathogenesis of neuropsychiatric disorders [38]. Previous reports also demonstrated that oxidative stress was activated during neuroinflammation [39]. The observation was confirmed by our findings, which indicated that several markers reflecting the status of oxidative stress, including MDA, SOD and GSH-pX, were evidently changed by LPS administration, suggesting the presence of a higher oxidative response and a lower antioxidant status. Notably, PRMT1 knockout significantly alleviated LPS-induced oxidative stress via the expression of Nrf-2. Excessive studies have reported that Nrf-2 is involved in oxidative and inflammatory damage [21,40]. Here, we also found that suppressing Nrf-2 expression abolished PRMT1 knockout-attenuated inflammation and ROS generation in LPSstimulated AST. Therefore, PRMT1 could meditate Nrf-2 expression to influence LPS-induced inflammation and oxidative stress, which subsequently affected anxiety- and depression-like behaviors. In conclusion, our data illustrated that LPS exposure in mice resulted in neuroinflammatory and oxidative responses, and importantly the process could be restrained by the knockout of PRMT1 at least partly dependent on Nrf-2 signaling. Thereby, PRMT1 could be served as an important therapeutic target for developing novel and effective treatment to ameliorate depression in patients. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.032. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.032. References [1] E. Pettersson, et al., Common psychiatric disorders share the same genetic origin: a multivariate sibling study of the Swedish population, Mol. Psychiatry 21 (5) (2016) 717. [2] Y. Wang, et al., Influence of anti-depression therapy on inflammatory cytokines and quality of life in patients with acute coronary syndrome complicating depression, Int. J. Lab. Med. 38 (2017) 1365e1367. [3] A.L. Lopresti, et al., A review of peripheral biomarkers in major depression: the potential of inflammatory and oxidative stress biomarkers, Prog. Neuro Psychopharmacol. Biol. Psychiatry 48 (2014) 102e111. [4] Y. Dowlati, et al., A meta-analysis of cytokines in major depression, Biol.
Psychiatry 67 (2010) 446e457. [5] S.A. Hiles, et al., A meta-analysis of differences in IL-6 and IL-10 between people with and without depression: exploring the causes of heterogeneity, Brain Behav. Immun. 26 (2012) 1180e1188. [6] B.S.F. Mello, et al., Effects of doxycycline on depressive-like behavior in mice after lipopolysaccharide (LPS) administration, J. Psychiatr. Res. 47 (2013) 1521e1529. re, et al., Methyltransferase PRMT1 Is a [7] S. Benhenda, A. Ducroux, L. Rivie binding partner of HBx and a negative regulator of hepatitis B virus transcription, J. Virol. 87 (8) (2013) 4360e4371. [8] X. Zhao, et al., Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity, Genes Dev. 22 (2008) 640e653. [9] M. Le Romancer, et al., Regulation of estrogen rapid signaling through arginine methylation by PRMT1, Mol. Cell 31 (2008) 212e221. [10] Q. Sun, et al., PRMT1 upregulated by epithelial proinflammatory cytokines participates in COX2 expression in fibroblasts and chronic antigen-induced pulmonary inflammation, J. Immunol. (2015), 1402465. [11] M.L. Onozato, et al., Expression of DDAH and PRMT isoforms in the diabetic rat kidney; effects of angiotensin II receptor blocker, Diabetes 57 (2007). [12] W. Shao, et al., Suppression of neuroinflammation by astrocytic dopamine D2 receptors via aB-crystallin, Nature 494 (2013) 90. [13] Q. Ren, et al., Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress, Proc. Natl. Acad. Sci. 113 (2016) E1944eE1952. [14] Q. Ren, et al., BDNFeTrkB signaling in the nucleus accumbens shell of mice has key role in methamphetamine withdrawal symptoms, Transl. Psychiatry 5 (2015), e666. [15] C. Yang, et al., R-ketamine: a rapid-onset and sustained antidepressant without psychotomimetic side effects, Transl. Psychiatry 5 (2015), e632. [16] M. Li, et al., Lentivirus-mediated interleukin-1b (IL-1b) knock-down in the hippocampus alleviates lipopolysaccharide (LPS)-induced memory deficits and anxiety-and depression-like behaviors in mice, J. Neuroinflammation 14 (2017) 190. [17] M.P. Kaster, et al., Depressive-like behavior induced by tumor necrosis factora in mice, Neuropharmacology 62 (2012) 419e426. [18] J. Zhang, et al., Antidepressant effects of TrkB ligands on depression-like behavior and dendritic changes in mice after inflammation, Int. J. Neuropsychopharmacol. 18 (2015). [19] S.A. Villeda, et al., Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice, Nat. Med. 20 (2014) 659. [20] T.V. Lipina, et al., Disrupted-in-schizophrenia-1 Gln31Leu polymorphism results in social anhedonia associated with monoaminergic imbalance and reduction of CREB and b-arrestin-1, 2 in the nucleus accumbens in a mouse model of depression, Neuropsychopharmacology 38 (2013) 423. [21] J.P. Guilloux, et al., Molecular evidence for BDNF-and GABA-related dysfunctions in the amygdala of female subjects with major depression, Mol. Psychiatry 17 (2012) 1130. [22] M.V. Doucet, et al., Small-molecule inhibitors at the PSD-95/nNOS interface have antidepressant-like properties in mice, Neuropsychopharmacology 38 (2013) 1575. [23] S.H. Seo, et al., Fisetin inhibits TNF-a-induced inflammatory action and hydrogen peroxide-induced oxidative damage in human keratinocyte HaCaT cells through PI3K/AKT/Nrf-2-mediated heme oxygenase-1 expression, Int. Immunopharmacol. 29 (2015) 246e253. [24] M. Ma, et al., Antidepressant effects of combination of brexpiprazole and fluoxetine on depression-like behavior and dendritic changes in mice after inflammation, Psychopharmacology 234 (2017) 525e533. [25] S.M. Clark, et al., Dissociation between sickness behavior and emotionality during lipopolysaccharide challenge in lymphocyte deficient Rag2/ mice, Behav. Brain Res. 278 (2015) 74e82. [26] L. Ge, et al., Resveratrol abrogates lipopolysaccharide-induced depressive-like behavior, neuroinflammatory response, and CREB/BDNF signaling in mice, Eur. J. Pharmacol. 768 (2015) 49e57. [27] Y. Ohgi, et al., Effects of antidepressants on alternations in serum cytokines and depressive-like behavior in mice after lipopolysaccharide administration, Pharmacol. Biochem. Behav. 103 (2013) 853e859. [28] Z. Wang, et al., The effects of curcumin on depressive-like behavior in mice after lipopolysaccharide administration, Behav. Brain Res. 274 (2014) 282e290. [29] A.K. Walker, et al., NMDA receptor blockade by ketamine abrogates lipopolysaccharide-induced depressive-like behavior in C57BL/6J mice, Neuropsychopharmacology 38 (2013) 1609. [30] X. Deng, et al., Lipolysaccharide-induced neuroinflammation is associated with Alzheimer-like amyloidogenic axonal pathology and dendritic degeneration in rats, Adv. Alzheimer's Dis. 3 (2014) 78. [31] B. Cha, et al., Protein arginine methyltransferases (PRMTs) as therapeutic targets, Expert Opin. Ther. Targets 16 (2012) 651e664. [32] Q. Sun, et al., Upregulated protein arginine methyltransferase 1 by IL-4 increases eotaxin-1 expression in airway epithelial cells and participates in antigen-induced pulmonary inflammation in rats, J. Immunol. (2012), 1102635. [33] D.T. Skelly, et al., A systematic analysis of the peripheral and CNS effects of systemic LPS, IL-1b, TNF-a and IL-6 challenges in C57BL/6 mice, PLoS One 8 (2013), e69123. [34] Y.Y. Zhang, et al., Atorvastatin attenuates the production of IL-1b, IL-6, and
H. Liu et al. / Biochemical and Biophysical Research Communications 518 (2019) 430e437 TNF-a in the hippocampus of an amyloid b1-42-induced rat model of Alzheimer's disease, Clin. Interv. Aging 8 (2013) 103. [35] D.M. Norden, et al., Sequential activation of microglia and astrocyte cytokine expression precedes increased iba-1 or GFAP immunoreactivity following systemic immune challenge, Glia 64 (2016) 300e316. [36] D.I. Kim, et al., PRMT1 and PRMT4 regulate oxidative stress-induced retinal pigment epithelial cell damage in SIRT1-dependent and SIRT1-independent manners, Oxidative med. cell. longev. (2015) 2015. [37] C. Savoia, et al., Countervailing vascular effects of rosiglitazone in high cardiovascular risk mice: role of oxidative stress and PRMT-1, Clin. Sci. 118
437
(2010) 583e592. [38] D. Ben-Shachar, et al., Dopamine toxicity involves mitochondrial complex I inhibition: implications to dopamine-related neuropsychiatric disorders, Biochem. Pharmacol. 67 (2004) 1965e1974. [39] N. Bakunina, et al., Immune mechanisms linked to depression via oxidative stress and neuroprogression, Immunology 144 (2015) 365e373. [40] C.H. Kang, et al., 5-Hydroxy-3, 6, 7, 8, 30 40 -hexamethoxyflavone inhibits nitric oxide production in lipopolysaccharide-stimulated BV2 microglia via NF-kB suppression and Nrf-2-dependent heme oxygenase-1 induction, Food Chem. Toxicol. 57 (2013) 119e125.