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RIPK1 axis
Biomedicine & Pharmacotherapy 125 (2020) 109818
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Inhibitory effects of JQ1 on listeria monocytogenes-induced acute liver injury by blocking BRD4/RIPK1 axis
T
Zhao Qiana,1, Wang Shuyingb,1, Ding Ranranc,* a
Department of Emergency, Hebei General Hospital, Shijiazhuang, Hebei, 050051, China Department of Emergency, Shanxian Central Hospital, Shanxian County, Shandong Province, 274300, China c Department of Intensive Care Unit, Jining NO.1 People's Hospital, Jining City, Shandong Province, 272000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Listeria monocytogenes Acute liver injury BRD4 JQ1 RIPK1
Listeria monocytogenes (LM) is a facultative intracellular bacterium that causes septicemia-associated acute hepatic injury. However, the pathogenesis of this process is still unclear, and there is still a lack of effective therapeutic strategy for the treatment of LM-induced liver injury. In this study, we attempted to explore the effects of necroptosis on bacterial-septicemia-associated hepatic disease and to explore the contribution of JQ1, a selective BRD4 inhibitor, to the suppression of necroptosis and inhibition of LM-triggered hepatic injury. The results indicated that hepatic BRD4 was primarily stimulated by LM both in vitro and in vivo, along with significantly up-regulated expression of receptor-interacting protein kinase (RIPK)-1, RIPK3, and p-mixed lineage kinase-like (MLKL), showing the elevated necroptosis. However, JQ1 treatment and RIPK1 knockout were found to significantly alleviate LM-induced acute liver injury. Histological alterations and cell death in hepatic samples in LM-infected mice were also alleviated by JQ1 administration or RIPK1 deletion. However, JQ1-improved hepatic injury by LM was abrogated by RIPK1 over-expression, suggesting that the protective effects of JQ1 took place mainly in an RIPK1-dependent manner. In addition, LM-evoked inflammatory response in liver tissues were also alleviated by JQ1, which was similar to the findings observed in mice lacking RIPK1. The anti-inflammatory effects of JQ1 were diminished by RIPK1 over-expression in LM-infected mice. Finally, both in vivo and in vitro experiments suggested that JQ1 dramatically improved hepatic mitochondrial dysfunction in LMinjected mice, but this effect was abolished by RIPK1 over-expression. In conclusion, these results indicated that suppressing BRD4 by JQ1 could ameliorate LM-associated liver injury by suppressing necroptosis, inflammation, and mitochondrial dysfunction by inhibiting RIPK1.
1. Introduction Listeria monocytogenes (LM), one of the best-studied pathogens in immunology and microbiology, has been reported to cause severe and life-threatening infections primarily among human [1,2]. Experimental infections with LM brought profound insights into the cell biological and immunological responses for the infected individuals [3]. Increasing studies have suggested that septicemia resulted from LM infection, and it is a key to induce acute liver injury [4,5]. Acute liver injury is a complex and life-threatening syndrome, leading to sudden or massive hepatic cell death and dysfunction, and eventually multiple organ failure [6]. Dysregulated inflammatory response is a pivotal component of innate immunity that contributes to the initiation and progression of acute hepatic injury [7]. Acute liver injury can also be caused by cell death [8]. Necroptosis is a newly reported form of cell
death, an inflammatory form of necrotic cell death, and its process is tightly modulated, like that of apoptosis [9]. The role of the receptorinteracting protein kinase 1 (RIPK1) signaling pathway in the progression of necroptosis has been widely investigated [10]. The activation of the RIPK1/RIP3 complex could recruit and phosphorylate the down-streaming MLKL, reported as an executor of necroptosis [11]. Suppressing RIPK1/RIPK3/MLKL activation is an effective means of inhibiting necroptosis and healing acute liver injury under various stimuli conditions [12]. Although the effects of RIPK1/RIPK3/MLKL signaling have been investigated, if there are other potential factors involved in necroptosis that contribute to acute liver injury, they are still largely unclear. Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal (BET) protein family. BRD4 has been proposed as a potential therapeutic target for patients with fibrotic
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Corresponding author. E-mail address: [email protected] (D. Ranran). 1 Qian Zhao & Shuying Wang are the co-first authors. https://doi.org/10.1016/j.biopha.2020.109818 Received 21 October 2019; Received in revised form 12 December 2019; Accepted 15 December 2019 0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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saturated incubator at 37 °C. Listeriolysin-O (LLO) (GenWay Biotech, Inc., USA) derived from LM was used as inducers to estimate a series of responses induced by LM. To reactivate RIPK1 expression in cells, AdRIPK1 were transfected into KCs in diluted medium at a multiplicity of infection of 50 for 24 h [18].
complications [13,14]. Recently, it has been suggested that BRD4 may positively modulate necroptosis and suppress BRD4 via its selective inhibitor JQ1 and so inhibit MLKL-meditated necroptosis. This has been found to contribute to the inhibition of inflammatory response syndrome in rodent animals [15]. Given the critical role of BRD4 in regulating hepatic disease and necroptosis, we hypothesize that targeting BRD4 might be an effective means of treating septicemia-related acute liver injury. In this study, we attempted to investigate: (i) whether changes in BRD4 expression were involved in LM-induced acute hepatic injury; (ii) RIPK1-regulated necroptosis was involved in the disease progression; and (iii) whether reducing BRD4 expression via JQ1 had any protective effects against septicemia-associated acute hepatic injury and whether it exerted those effects through suppression of necroptosis as regulated by the RIPK1 signaling pathway.
2.3. Cell viability analysis MTT analysis (KeyGen Biotech, China) was used to determine the cell viability according to the manufacturer’s protocols. 20 μL of MTT solution was added to cells after treatments. After incubation at 37 °C for another 4 h, the supernatants were removed and DMSO (KeyGen Biotech) was added to each well for 10 min. Then, the absorbance was read at 490 nm using a microplate reader. 2.4. Biochemical parameters
2. Materials and methods Tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-18, IL-1β and monocyte chemotactic protein-1 (MCP-1) concentrations in liver samples were measured using commercially available ELISA kits (BD Bioscience, USA) according to the manufacturer’s protocols. Serum alanine aminotransferase (ALT), aspartat transaminase (AST) and alkaline phosphatase (AKP) activities were measured using commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s protocols. Mitochondria were isolated using the MitoCheck Mitochondrial Isolation Kit (Cayman Chemical, USA). Complexe I and IV activities were measured using respective commercial kits (Cayman Chemical) according to the manufacturer’s instructions.
2.1. Animals and treatments All animal experiments were performed in accordance with protocols for Animal Research Committee of the Hebei General Hospital (Shijiazhuang, China), and the National Institute of Health (NIH) guidelines for the care and use of laboratory animals. Male, 6–8 weeks old, weighing 18−22 g, wild type C57BL/6 mice (WT) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). Male, RIPK1 knockout (RIPK1-KO) mice (6–8 weeks old, weighing 18−22 g) with a C57BL/6 background were purchased from Cyagen US Inc. (Suzhou, China). All mice were acclimatized for 1 week before performing the experiments. The mice were housed in a constant temperature and humidity environmental condition (24 ± 2 °C, 52 ± 5% relative humidity, 12 h light-dark cycle) with free access to food and water. The mice were divided into 8 groups (n = 8/group): (1) WT/Ctrl; (2) RIPK1KO/Ctrl; (3) JQ1/Ctrl (without LM infection); (4) WT/LM (L. monocytogenes infection; ATCC 15313); (5) RIPK1KO/LM; (6) JQ1/LM; (7) JQ1+AdCtrl/LM; (8) JQ1+AdRIPK1/LM. The 50 mg/kg of JQ1 solution (MedChemExpress, USA) was treated to mice by intraperitoneal injection 3 times a week for 4 weeks. The concentration of JQ1 chosen for animals was referred to previous study [16]. For hepatic-specific gene overexpression in JQ1-treated mice, adenoviral vectors that expressed β-galactosidase (Ad-Ctrl) and RIPK1 (Ad-RIPK1) (5 × 109 plaque-forming units) were injected into JQ1-treated mice through the jugular vein 24 h before LM infection according to previous study [17]. At the end of pre-treatment of JQ1, one single injection of LM (1 × 106cfu) was subjected to mice by intraperitoneal injection. After LM infection for 0, 4, 8, 12, 16 or 24 h, mice were sacrificed, and liver samples were collected for further analysis.
2.5. Transmission electron microscopy (TEM) Liver samples were fixed in 2.5% glutaraldehyde, followed by incubation with phosphoric acid buffer and fixed in 1% osmium acid at 4 °C in the dark. The post fixation process was performed using 2% osmium tetroxide. After being dehydrated in a graded series of ethanol and propylene oxide, the tissues were embedded with epoxy resin. Sections (50–70 nm) were cut for staineing using uranyl acetate and lead citrate. Images were viewed using a Hitachi 7600 TEM (USA). 2.6. Quantitative real-time polymerase chain reaction (RT-qPCR) assays As for RT-qPCR analysis, total RNA was isolated from hepatic samples using TRIzol Reagent (Invitrogen) following the manufacturer’s instructions. The obtained total RNA was used to prepare cDNA with RevertAidTM First Strand cDNA Synthesis Kit (Fermentas Life Sciences, USA) according to the manufacturer’s protocols. The cDNA was then amplified by RT-qPCR using a thermocycler (Lightcycler®Nano, Roche, USA) with SYBR Green detection system (Roche). Primers used in the study were synthesized by Sangon Biotech (Guangzhou, China), and the listed in Supplementary table 2. Results were expressed as the relative mRNA level of specific target gene, and were normalized to GAPDH levels, as obtained using the ΔΔCT method.
2.2. Cell isolation and treatments The male, 8–10 weeks old, healthy C57BL/6 mice were anesthetized using diethyl ether. Then, the liver tissues were carefully excised. The liver tissues were then cut into pieces, and incubated with pronase E and collagenase type I. Then, the mixture was cultured and shaken at 37 °C for 30 min. After that, DNase I (Sigma Aldrich, USA) was added to the mixture for co-incubation for 20 min. Subsequently, stainless steel mesh was used for filtration before washing the cells and dissolving erythrocyte with trisammoniachloride. Gradient centrifugation was conducted using 30–70% Percoll and maintained in uncoated culture flasks containing RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, GIBCO Corporation, USA) and 1 × 105 U/L streptomycin sulfate at a concentration of 1 × 105/mL for 4–6 h. The cells that not attached were removed. The obtained cells were then used for further analysis. The obtained KCs were cultured in RPMI 1640 medium (GIBCO Corporation, USA) supplemented with 10% fetal bovine serum and 1 × 105 U/L streptomycin sulfate in a 5% CO2, water-
2.7. Cellular terminal deoxynucleotidyl transferase (TdT)-mediated dUTPbiotin nick end labeling (TUNEL) staining Apoptotic cells were calculated using a commercially available kit (Apoptosis Detection kit, Takara, Japan) following the manufacturer’s instructions. The percentage of TUNEL-positive cells was analyzed and quantified using an automated digital image analyzer. 2.8. Western blot assay Protein was extracted from cells or liver tissues using RIPA buffer (Beyotime, Nanjing, China). 20−50 μg protein were separated by 10% 2
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samples were observed in mice infected with LM (Fig. 1H). Together, the results given above indicate that LM infection could up-regulate BRD4 expression and necroptosis via RIPK1/RIPK3/MLKL, which might be involved in the progression of acute liver injury.
SDS-PAGE, and were then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% dry milk, followed by incubation with primary antibodies overnight at 4 °C (Supplementary Table S1). Then, the membranes were incubated with secondary antibodies (Beyotime). The antibody/antigen complexes were determined with the ECL system (ThermoFisher Scientific, USA), and the optical densities of each band were analyzed by the ImageJ software.
3.2. JQ1 alleviates acute liver injury through suppressing RIPK1 activation The results given above indicated that BRD4 and the RIPK1/RIPK3/ MLKL signaling pathways were associated with LM-induced acute liver injury. As previously indicated, changes in BRD4 expression were linked to necroptosis [15]. We here used RIPK-1 knockout mice with and without JQ1 pretreatment and LM infection to determine if BRD4 suppression that takes place via its inhibitor JQ1 was effective for the suppression of acute liver injury and assess the underlying molecular mechanisms. At first, liver damage was examined by serum levels of AST, ALT, and AKP in WT or RIPK1-KO mice. As shown in Fig. 2A, serum AST, ALT, and AKP levels were markedly higher in WT-mice with LM infection than in a control group of uninfected WT mice. However, these effects were significantly reduced in mice with RIPK1 knockout or JQ1 pre-treatment. Moreover, to confirm whether JQ1 could effectively diminish the level of RIPK1-associated necroptosis, adenoviral vectors (Ad) over-expressing RIPK1 (AdRIPK1) and the negative control vectors (AdCtrl) were injected into the JQ1-treated mice 24 h before LM-induced acute liver injury. Administration of AdRIPK1 negated the protective role of JQ1 in reducing AST, ALT, and AKP levels. H&E and TUNEL staining showed that, after LM infection, WT-mice exhibited obvious necroptosis and histological changes in hepatic tissues, with significant up-regulation of cell death. These phenomena were alleviated to a great extent by RIPK1 knockout and JQ1 pre-treatment individually. In comparison, over-expressing RIPK1 abrogated the protective effects of JQ1 on histological alterations and cell death (Fig. 2B and C). There was considerably more RIPK1, RIPK3, p-MLKL, and BRD4 expression in groups with LM infection than in the WT/Ctrl group. JQ1 pre-treatment and RIPK1 deficiency both rendered the augmentation of RIPK1, RIPK3, pMLKL, and BRD4 less pronounced, demonstrating an inhibitory effect of JQ1 on RIPK1-associated necroptosis in acute liver injury by LM infection. However, promoting RIPK1 expression significantly abrogated the role of JQ1 in the repression of these signals (Fig. 2D–H). These findings above suggested that suppressing BRD4 expression by JQ1 could attenuate acute liver injury through repressing RIPK1 expression.
2.9. Immunohistochemical(IHC) analysis Hepatic tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections (5-μm thickness) were affixed to slides, deparaffinized, and then were subjected to hematoxylin and eosin (H&E) staining. For IHC staining, hepatic sections were incubated with primary antibody F4/80 (1:150 dilutions, Abcam, USA) overnight at 4 °C. After washing, the sections were incubated with HRP-conjugated secondary antibody (Beyotime), followed by 3,3′-diaminobenzidine (DAB) (Sigma Aldrich, USA) incubation. Then, the representative images were obtained using a light microscope. As for apoptosis in liver, the paraffin-embedded hepatic sections were stained by TUNEL assay (KeyGen Biotech, Nanjing, China) to calculate in vivo cell death according to the manufacturer’s protocols. TUNEL-stained liver tissues were visualized under a light microscope. 2.10. Mitochondrial membrane potential and mitochondrial permeability transition pore (mPTP) calculation The mitochondrial membrane potential was determined using the JC-1 probe (Beyotime) according to the manufacturer’s protocols. mPTP opening was determined as previously indicated using tetramethylrhodamine ethyl ester. The relative mPTP opening was calculated as a ratio to that of the control group [19]. 2.11. Mitochondrial DNA calculation Mitochondrial DNA was quantified according to previous study [20]. 2.12. Statistical analysis All results are presented as the mean ± standard error of the mean (SEM.). Statistical comparisons were performed using one-way analysis of variance (ANOVA), difference between two groups was analyzed using Student’s two-tailed t-test. P value < 0.05 for the difference was considered statistically significant. Details for materials and methods used in the study were exhibited in Supplementary part.
3.3. JQ1 alleviates hepatic inflammation in LM-infected mice Inflammation plays a critical role in regulating acute liver injury, which is associated with necroptosis [21]. Here, we established that pro-inflammatory cytokines and chemokines, including TNF-α, IL-6, IL1β, IL-18, MCP-1, and MCP-2, were highly induced by LM in liver tissue samples; however, the elevation of these factors triggered by LM was markedly abrogated by JQ-1 pre-treatment and by RIPK1 deletion. RIPK1 over-expression abolished the anti-inflammatory effects of JQ1, contributing to the severity of liver injury (Fig. 3A and B). In addition, hepatic p-IκBα and p-NF-κB expression levels stimulated by LM were greatly reduced by JQ-1 administration and RIPK1 deletion. In comparison, RIPK1 over-expression markedly abrogated the role of JQ1 in blocking IκBα/NF-κB signaling (Fig. 3C and D). Moreover, JQ1 pretreatment and RIPK1 knockout significantly reduced inflammatory cell recruitment in liver tissues from LM-infected mice, as evidenced by the down-regulated levels of expression of CD68. However, these effects regulated by JQ1 were markedly abrogated by RIPK1 over-expression (Fig. 3E). These results established that JQ1-suppressed inflammation in acute liver injury by LM infection took place mainly in an RIPK1dependent manner.
3. Results 3.1. BRD4/RIPK1 activation is associated with the progression of acute liver injury To calculate the effects of BRD4 on acute liver injury, changes in its expression were detected in vitro and in vivo. As shown in Fig. 1A and B, LLO-infected KCs showed significantly up-regulated expression of BRD4 from mRNA and protein levels compared to the Ctrl group. Meanwhile, the levels of expression of necroptosis markers, such as RIPK1, RIPK3, and p-MLKL, were found to be markedly induced by LLO stimulation in a dose-dependent manner (Fig. 1C). In vivo, we also found that levels of BRD4 expression in hepatic samples were markedly more induced by LM infection than in the control group (Fig. 1D and E). LM-infected mice also showed significantly increased expression of RIPK1, RIPK3, and p-MLKL in liver tissues (Fig. 1F). TUNEL staining showed that LLO treatment led to detectable cell death in KCs (Fig. 1G). Consistently, significantly increased numbers of TUNEL-positive cells in hepatic
3.4. JQ1 improves mitochondrial function by suppressing RIPK1 signaling The condition of mitochondria plays a critical role in meditating 3
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Fig. 1. LM infection up-regulates BRD4 expression in hepatic tissues of mice. (A) RT-qPCR and (B) western blot analysis for BRD4 in KCs treated with LLO (62.5, 125 or 250 ng/ml) for 8 h. (C) Western blot results for RIPK1, RIPK3 and p-MLKL in KCs incubated with LLO (62.5, 125 or 250 ng/ml) for 8 h.(D) RT-qPCR and (E) western blot analysis for BRD4 in liver tissues of LM-infected mice for the indicated time (0, 4, 8, 12, 16 or 24 h). (F) Western blot results for RIPK1, RIPK3 and pMLKL in hepatic samples of LM-infected mice for the shown time points (0, 4, 8, 12, 16 or 24 h). (G) KCs were exposed to LLO (62.5, 125 and 250 ng/ml) for 8 h, followed by apoptosis measurement using TUNEL staining. (H) TUNEL staining of liver sections from LM-infected mice at the indicated time. Data were expressed by means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001vs the Con group.
opposite results were detected in the change of mPTP opening in KCs as shown in Fig. 4G. Then, JC-1 staining further established that LLO-incubated KCs had significantly impaired mitochondrial membrane potential, which was rescued by JQ1 incubation or RIPK1 deletion. The protective role of JQ1 in improving mitochondrial membrane potential took place in an RIPK3-dependent manner, and similar results were detected in the mtDNA levels (Fig. 4J and K). Therefore, RIPK1 suppression was also required for improving mitochondrial function exerted by JQ1.
RIPK1-regulated necroptosis [22]. To further investigate the molecular mechanism underlying JQ1-modulated RIPK1 inactivation and suppression of necroptosis in LM-induced acute liver injury, mitochondrial function was evaluated in vivo and in vitro. As shown in Fig. 4A, TEM suggested that LM infection caused mitochondrial impairments in liver sections. JQ1 administration-improved mitochondrial was, however, abolished by RIPK1 over-expression in liver of LM-infected mice (Fig. 4A). We then found that hepatic ATP contents and mitochondrial membrane potential were significantly reduced by LM infection, and these effects were rescued by JQ1 administration or RIPK1 knockout. Over-expression of RIPK1 markedly abrogated the protective effects of JQ1 against mitochondrial function (Fig. 4B and C). Then, Western blot analysis was used to calculate the protein expression levels of signals associated with mitochondrial function, including DRP1, MFF, FIS1, PGAM5, and CypD [23,24]. LM-infected WT mice showed significantly higher levels of expression of DRP1, MFF, FIS1, PGAM1, and CypDin liver tissues than in the control, and the promotion of these proteins was attenuated by JQ1 and RIPK1 deletion. The inhibitory role of JQ1 in suppressing these molecules was found to operate in a RIPK1-dependent manner (Fig. 4D). To confirm the effects of JQ1 against mitochondrial dysfunction, the in vitro study was then performed using KCs isolated from the WT or RIPK1-KO mice. At first, MTT results suggested that the cell viability was visibly reduced by LLO stimulation, but was restored near to normal levels by JQ1 incubation or RIPK1 knockout. However, RIPK1 over-expression through AdRIPK1 transfection resulted in the reduction of cell viability (Fig. 4E). Consistently, ATP contents, Complex I activity, and Complex IV activity were found to be lower in LLO-incubated KCs, and it returned to near-normal levels due to JQ1 pre-treatment or RIPK1 deletion. RIPK1 over-expression markedly abolished the effects of JQ1 on the improvement of ATP, Complex I, and Complex IV after LLO stimulation (Fig. 4F, H and I). The
4. Discussion In this study, we found levels of BRD4 expression to be markedly upregulated in mice with acute liver injury induced by LM. The RIPK1/ RIPK3/MLKL signaling pathway was also activated after LM infection, contributing to the necroptosis and subsequent hepatic damage. RIPK1 genetic deletion attenuated necroptosis, inflammation, and mitochondrial dysfunction in hepatic tissues from LM-infected mice. Notably, suppressing BRD4 with JQ1 markedly reduced RIPK1 activation, inactivated RIPK3/MLKL signaling, repressed inflammatory response, and alleviated mitochondrial disturbance, which demonstrated the protective effects of JQ1 against acute liver injury associated with septicemia. Increasing RIPK1 expression markedly abrogated the protective effects of JQ1, indicating that JQ1-improved acute liver injury was largely RIPK1 dependent. To our knowledge, this is the first evidence that the effects of JQ1 on LM-induced necroptosis, which was focused on RIPK1 signaling. BRDs are crucial epigenetic “reader” proteins, which specifically recognize acetylated-lysine residues in nucleosomal histones, thus facilitating the recruitment of transcriptional proteins to chromatin [13,14]. The BRD4 inhibitor JQ1 was found to have anti-inflammatory, 4
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Fig. 2. JQ1 improves acute liver injury through suppressing RIPK1 activation after LM infection for 24 h. (A) Serum ALT, AST and AKP levels were assessed. (B) Up panel, H&E staining of hepatic sections; down panel, TUNEL staining of liver specimens. (C) Quantification of TUNEL-positive levels in hepatic samples. (D–H) RIPK1, RIPK3, p-MLKL and BRD4 protein levels in liver tissue were measured by western blotting analysis. Data were expressed by means ± SEM. **p < 0.01 and ***p < 0.001vs the Ctrl/WT group; +p < 0.05, ++p < 0.01 and +++p < 0.001vs the LM/WT group; #p < 0.05 and ##p < 0.01vs the LM/JQ1+AdCtrl group.
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Fig. 3. JQ1 alleviates hepatic inflammation in24 h of LM-infected mice. (A) ELISA analysis for TNF-α, IL-6, IL-1β, IL-18 and MCP-1 in liver samples. (B) RT-qPCR analysis for TNF-α, IL-6, IL-1β, IL-18, MCP-1 and MCP-2 in hepatic tissues. (C and D) Western blot analysis was used to determine p-IκBα and p-NF-κB protein expression levels in liver samples. (E)IHC staining for F4/80 in hepatic sections. Data were expressed by means ± SEM.*p < 0.05, **p < 0.01 and ***p < 0.001vs the Ctrl/WT group; +p < 0.05 and++p < 0.01vs the LM/WT group; #p < 0.05 and ##p < 0.01vs the LM/JQ1+AdCtrl group.
which was similar to the inhibitory role of RIPK1 knockout. Notably, over-expression of RIPK1 abrogated the anti-inflammatory effects of JQ1 in LM-challenged mice. These findings indicated that the JQ1suppressed inflammatory response was largely RIPK1-dependent. MLKL could lead to mitochondria fission through PGAM5 and DRP1 [33]. PGAM5 plays a significant role in regulating mitochondrial homeostasis and various necrotic death pathways [34]. The downstreaming executive events of necroptosis are RIPK3-induced activation of PGAM5 and mPTP opening [35]. Functional analysis clearly indicated that the activation of RIPK3 increased PGAM5 expression, which subsequently enhanced CypD expression and caused cells to undergo necroptosis by promoting the opening of mPTP [24]. Mitochondrial dysfunction has been linked to various hepatic diseases, including acute liver injury induced by LM and lipopolysaccharide [36]. PGAM5 can regulate mitochondrial fission to control mitochondrial dynamics [37]. DRP1, a large GTPase, plays an essential role in meditating the mitochondrial fission in mammalian cells [38]. The RIPK1/RIPK3 complex can initiate mitochondrial fission, which is involved in the promotion of inflammation [39]. PGAM5 is strictly necessary for concanavalinA (ConA)-induced hepatic necroptosis and liver injury [40]. In our study, we found for the first time that JQ1 treatment markedly alleviated mitochondrial impairment and dysfunction in livers of LM-infected mice and in LLO-exposed KCs, as proved by the reduced levels of expression of PGAM5, CypD, DRP1, MFF, and FIS1. MFF and FIS1 are hallmarks of mitochondrial fission, playing an essential role in inducing the disorder of mitochondria [23]. However, over-expression of RIPK3 was found to markedly abrogate the role of JQ1 in improving mitochondrial function, further indicating that JQ1-
anti-fibrotic, and anti-tumor effects [14,25,26]. More recently, changes in BRD4 expression were reported to be associated with necroptosis by regulating MLKL activation [14]. Necroptosis, a form of cell death, critically depends on RIPK3/MLKL signaling activation regulated by RIPK1 [11,12]. Acute liver injury is a life-threatening syndrome due because of its massive inflammation and cell death. Suppressing necroptosis is an effective means of treating acute liver failure as induced by d-galactosamine or lipopolysaccharide [27]. In this study, we further confirmed the effects of BRD4 on the necroptosis progression in mice with acute liver injury driven by LM. First, JQ1 treatment alleviated RIPK3 and MLKL activation in the livers of LM-infected mice, which was similar to the effects of RIPK1 knockout. The programmed necroptosis regulated by RIPK3 has served as an inflammatory form of cell death with critical functions in pathogeninduced and sterile inflammation [28]. RIPK3-regulated necrotic cell death promotes systematic inflammation and mortality [11]. Fatal septicemia is closely associated with systemic inflammatory response syndrome. This process is attributed to the excessive release of proinflammatory cytokines (TNF-α, IL-6, IL-18, and IL-1β) and chemokines (MCP-1 and MCP-2) [29,30]. In our study, we confirmed that LM infection resulted in inflammatory response in hepatic tissues, as evidenced by the significantly increased expression of TNF-α, IL-6, IL-18, IL-1β, MCP-1, and MCP-2 by activating NF-κB pathway. NF-κB is a pivotal signaling factor involved in inducing the secretion of pro-inflammatory factors [31]. BRD4 suppression by JQ1 is also effective for the blockage of NF-κB pathway, subsequently reducing the production of inflammatory molecules [32]. Consistently, JQ1 treatment markedly abrogated LM-induced inflammation by repressing NF-κB activation,
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Fig. 4. JQ1 improves mitochondrial function by suppressing RIPK1 signaling after LM infection for 24 h. (A) TEM for liver samples. The red asterisk referred the impaired mitochondrial. (B) ATP contents in hepatic tissues. (C) Mitochondrial membrane potential(Ψm) was measured. (D) Western blot analysis of signals associated with mitochondrial function, including DRP1, MFF, FIS1, PGAM5 and CypD in liver tissues. (E–K) KCs isolated from the indicated groups of mice were pretreated with JQ1 for 24 h, and then transfected with AdRIPK1 for another 24 h, followed by exposure to LLO (250 ng/ml) for final 8 h. Then, all cells were harvested for the following studies. (E) Cell viability was measured using MTT analysis. (F) ATP contents in KCs were tested. (G) Evaluation of mPTP opening. (H) Complex I activity and (I) Complex IV activity in cells were assessed. (J) Mitochondrial membrane potential (Ψm) was measured using JC-1 staining. (K) Calculation of cellular mtDNA levels.Data were expressed by means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001vs the Ctrl/WT group; +p < 0.05 and++p < 0.01vs the LM/WT or LLO/WT group; #p < 0.05 and ##p < 0.01vs the LM/JQ1+AdCtrlor LLO/JQ1+AdCtrl group.
Conflict of interest
alleviated acute hepatic injury was RIPK3-dependent. In conclusion, the results in this study highlighted the critical role of necroptosis in acute liver injury as regulated by the RIPK1/BRD4 signaling pathway. These findings established that BRD4 inhibition by JQ1 could ameliorate necroptosis, inflammation, and mitochondrial dysfunction mainly through RIPK1 inhibition, consequently repressing septicemia-associated acute hepatic injury. BRD4 inhibitor might be an effective approach for the treatment of acute hepatic disease. Nevertheless, understanding of JQ1-induced RIPK1 inactivation is still poor. Further study is required to render our understanding more comprehensive.
The authors see no any conflict of interest in this study. Acknowledgments We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2020.109818. 7
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Inhibitory effects of JQ1 on listeria monocytogenes-induced acute liver injury by blocking BRD4/RIPK1 axis
T
Zhao Qiana,1, Wang Shuyingb,1, Ding Ranranc,* a
Department of Emergency, Hebei General Hospital, Shijiazhuang, Hebei, 050051, China Department of Emergency, Shanxian Central Hospital, Shanxian County, Shandong Province, 274300, China c Department of Intensive Care Unit, Jining NO.1 People's Hospital, Jining City, Shandong Province, 272000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Listeria monocytogenes Acute liver injury BRD4 JQ1 RIPK1
Listeria monocytogenes (LM) is a facultative intracellular bacterium that causes septicemia-associated acute hepatic injury. However, the pathogenesis of this process is still unclear, and there is still a lack of effective therapeutic strategy for the treatment of LM-induced liver injury. In this study, we attempted to explore the effects of necroptosis on bacterial-septicemia-associated hepatic disease and to explore the contribution of JQ1, a selective BRD4 inhibitor, to the suppression of necroptosis and inhibition of LM-triggered hepatic injury. The results indicated that hepatic BRD4 was primarily stimulated by LM both in vitro and in vivo, along with significantly up-regulated expression of receptor-interacting protein kinase (RIPK)-1, RIPK3, and p-mixed lineage kinase-like (MLKL), showing the elevated necroptosis. However, JQ1 treatment and RIPK1 knockout were found to significantly alleviate LM-induced acute liver injury. Histological alterations and cell death in hepatic samples in LM-infected mice were also alleviated by JQ1 administration or RIPK1 deletion. However, JQ1-improved hepatic injury by LM was abrogated by RIPK1 over-expression, suggesting that the protective effects of JQ1 took place mainly in an RIPK1-dependent manner. In addition, LM-evoked inflammatory response in liver tissues were also alleviated by JQ1, which was similar to the findings observed in mice lacking RIPK1. The anti-inflammatory effects of JQ1 were diminished by RIPK1 over-expression in LM-infected mice. Finally, both in vivo and in vitro experiments suggested that JQ1 dramatically improved hepatic mitochondrial dysfunction in LMinjected mice, but this effect was abolished by RIPK1 over-expression. In conclusion, these results indicated that suppressing BRD4 by JQ1 could ameliorate LM-associated liver injury by suppressing necroptosis, inflammation, and mitochondrial dysfunction by inhibiting RIPK1.
1. Introduction Listeria monocytogenes (LM), one of the best-studied pathogens in immunology and microbiology, has been reported to cause severe and life-threatening infections primarily among human [1,2]. Experimental infections with LM brought profound insights into the cell biological and immunological responses for the infected individuals [3]. Increasing studies have suggested that septicemia resulted from LM infection, and it is a key to induce acute liver injury [4,5]. Acute liver injury is a complex and life-threatening syndrome, leading to sudden or massive hepatic cell death and dysfunction, and eventually multiple organ failure [6]. Dysregulated inflammatory response is a pivotal component of innate immunity that contributes to the initiation and progression of acute hepatic injury [7]. Acute liver injury can also be caused by cell death [8]. Necroptosis is a newly reported form of cell
death, an inflammatory form of necrotic cell death, and its process is tightly modulated, like that of apoptosis [9]. The role of the receptorinteracting protein kinase 1 (RIPK1) signaling pathway in the progression of necroptosis has been widely investigated [10]. The activation of the RIPK1/RIP3 complex could recruit and phosphorylate the down-streaming MLKL, reported as an executor of necroptosis [11]. Suppressing RIPK1/RIPK3/MLKL activation is an effective means of inhibiting necroptosis and healing acute liver injury under various stimuli conditions [12]. Although the effects of RIPK1/RIPK3/MLKL signaling have been investigated, if there are other potential factors involved in necroptosis that contribute to acute liver injury, they are still largely unclear. Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal (BET) protein family. BRD4 has been proposed as a potential therapeutic target for patients with fibrotic
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Corresponding author. E-mail address: [email protected] (D. Ranran). 1 Qian Zhao & Shuying Wang are the co-first authors. https://doi.org/10.1016/j.biopha.2020.109818 Received 21 October 2019; Received in revised form 12 December 2019; Accepted 15 December 2019 0753-3322/ © 2020 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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saturated incubator at 37 °C. Listeriolysin-O (LLO) (GenWay Biotech, Inc., USA) derived from LM was used as inducers to estimate a series of responses induced by LM. To reactivate RIPK1 expression in cells, AdRIPK1 were transfected into KCs in diluted medium at a multiplicity of infection of 50 for 24 h [18].
complications [13,14]. Recently, it has been suggested that BRD4 may positively modulate necroptosis and suppress BRD4 via its selective inhibitor JQ1 and so inhibit MLKL-meditated necroptosis. This has been found to contribute to the inhibition of inflammatory response syndrome in rodent animals [15]. Given the critical role of BRD4 in regulating hepatic disease and necroptosis, we hypothesize that targeting BRD4 might be an effective means of treating septicemia-related acute liver injury. In this study, we attempted to investigate: (i) whether changes in BRD4 expression were involved in LM-induced acute hepatic injury; (ii) RIPK1-regulated necroptosis was involved in the disease progression; and (iii) whether reducing BRD4 expression via JQ1 had any protective effects against septicemia-associated acute hepatic injury and whether it exerted those effects through suppression of necroptosis as regulated by the RIPK1 signaling pathway.
2.3. Cell viability analysis MTT analysis (KeyGen Biotech, China) was used to determine the cell viability according to the manufacturer’s protocols. 20 μL of MTT solution was added to cells after treatments. After incubation at 37 °C for another 4 h, the supernatants were removed and DMSO (KeyGen Biotech) was added to each well for 10 min. Then, the absorbance was read at 490 nm using a microplate reader. 2.4. Biochemical parameters
2. Materials and methods Tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-18, IL-1β and monocyte chemotactic protein-1 (MCP-1) concentrations in liver samples were measured using commercially available ELISA kits (BD Bioscience, USA) according to the manufacturer’s protocols. Serum alanine aminotransferase (ALT), aspartat transaminase (AST) and alkaline phosphatase (AKP) activities were measured using commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s protocols. Mitochondria were isolated using the MitoCheck Mitochondrial Isolation Kit (Cayman Chemical, USA). Complexe I and IV activities were measured using respective commercial kits (Cayman Chemical) according to the manufacturer’s instructions.
2.1. Animals and treatments All animal experiments were performed in accordance with protocols for Animal Research Committee of the Hebei General Hospital (Shijiazhuang, China), and the National Institute of Health (NIH) guidelines for the care and use of laboratory animals. Male, 6–8 weeks old, weighing 18−22 g, wild type C57BL/6 mice (WT) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). Male, RIPK1 knockout (RIPK1-KO) mice (6–8 weeks old, weighing 18−22 g) with a C57BL/6 background were purchased from Cyagen US Inc. (Suzhou, China). All mice were acclimatized for 1 week before performing the experiments. The mice were housed in a constant temperature and humidity environmental condition (24 ± 2 °C, 52 ± 5% relative humidity, 12 h light-dark cycle) with free access to food and water. The mice were divided into 8 groups (n = 8/group): (1) WT/Ctrl; (2) RIPK1KO/Ctrl; (3) JQ1/Ctrl (without LM infection); (4) WT/LM (L. monocytogenes infection; ATCC 15313); (5) RIPK1KO/LM; (6) JQ1/LM; (7) JQ1+AdCtrl/LM; (8) JQ1+AdRIPK1/LM. The 50 mg/kg of JQ1 solution (MedChemExpress, USA) was treated to mice by intraperitoneal injection 3 times a week for 4 weeks. The concentration of JQ1 chosen for animals was referred to previous study [16]. For hepatic-specific gene overexpression in JQ1-treated mice, adenoviral vectors that expressed β-galactosidase (Ad-Ctrl) and RIPK1 (Ad-RIPK1) (5 × 109 plaque-forming units) were injected into JQ1-treated mice through the jugular vein 24 h before LM infection according to previous study [17]. At the end of pre-treatment of JQ1, one single injection of LM (1 × 106cfu) was subjected to mice by intraperitoneal injection. After LM infection for 0, 4, 8, 12, 16 or 24 h, mice were sacrificed, and liver samples were collected for further analysis.
2.5. Transmission electron microscopy (TEM) Liver samples were fixed in 2.5% glutaraldehyde, followed by incubation with phosphoric acid buffer and fixed in 1% osmium acid at 4 °C in the dark. The post fixation process was performed using 2% osmium tetroxide. After being dehydrated in a graded series of ethanol and propylene oxide, the tissues were embedded with epoxy resin. Sections (50–70 nm) were cut for staineing using uranyl acetate and lead citrate. Images were viewed using a Hitachi 7600 TEM (USA). 2.6. Quantitative real-time polymerase chain reaction (RT-qPCR) assays As for RT-qPCR analysis, total RNA was isolated from hepatic samples using TRIzol Reagent (Invitrogen) following the manufacturer’s instructions. The obtained total RNA was used to prepare cDNA with RevertAidTM First Strand cDNA Synthesis Kit (Fermentas Life Sciences, USA) according to the manufacturer’s protocols. The cDNA was then amplified by RT-qPCR using a thermocycler (Lightcycler®Nano, Roche, USA) with SYBR Green detection system (Roche). Primers used in the study were synthesized by Sangon Biotech (Guangzhou, China), and the listed in Supplementary table 2. Results were expressed as the relative mRNA level of specific target gene, and were normalized to GAPDH levels, as obtained using the ΔΔCT method.
2.2. Cell isolation and treatments The male, 8–10 weeks old, healthy C57BL/6 mice were anesthetized using diethyl ether. Then, the liver tissues were carefully excised. The liver tissues were then cut into pieces, and incubated with pronase E and collagenase type I. Then, the mixture was cultured and shaken at 37 °C for 30 min. After that, DNase I (Sigma Aldrich, USA) was added to the mixture for co-incubation for 20 min. Subsequently, stainless steel mesh was used for filtration before washing the cells and dissolving erythrocyte with trisammoniachloride. Gradient centrifugation was conducted using 30–70% Percoll and maintained in uncoated culture flasks containing RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, GIBCO Corporation, USA) and 1 × 105 U/L streptomycin sulfate at a concentration of 1 × 105/mL for 4–6 h. The cells that not attached were removed. The obtained cells were then used for further analysis. The obtained KCs were cultured in RPMI 1640 medium (GIBCO Corporation, USA) supplemented with 10% fetal bovine serum and 1 × 105 U/L streptomycin sulfate in a 5% CO2, water-
2.7. Cellular terminal deoxynucleotidyl transferase (TdT)-mediated dUTPbiotin nick end labeling (TUNEL) staining Apoptotic cells were calculated using a commercially available kit (Apoptosis Detection kit, Takara, Japan) following the manufacturer’s instructions. The percentage of TUNEL-positive cells was analyzed and quantified using an automated digital image analyzer. 2.8. Western blot assay Protein was extracted from cells or liver tissues using RIPA buffer (Beyotime, Nanjing, China). 20−50 μg protein were separated by 10% 2
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samples were observed in mice infected with LM (Fig. 1H). Together, the results given above indicate that LM infection could up-regulate BRD4 expression and necroptosis via RIPK1/RIPK3/MLKL, which might be involved in the progression of acute liver injury.
SDS-PAGE, and were then transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% dry milk, followed by incubation with primary antibodies overnight at 4 °C (Supplementary Table S1). Then, the membranes were incubated with secondary antibodies (Beyotime). The antibody/antigen complexes were determined with the ECL system (ThermoFisher Scientific, USA), and the optical densities of each band were analyzed by the ImageJ software.
3.2. JQ1 alleviates acute liver injury through suppressing RIPK1 activation The results given above indicated that BRD4 and the RIPK1/RIPK3/ MLKL signaling pathways were associated with LM-induced acute liver injury. As previously indicated, changes in BRD4 expression were linked to necroptosis [15]. We here used RIPK-1 knockout mice with and without JQ1 pretreatment and LM infection to determine if BRD4 suppression that takes place via its inhibitor JQ1 was effective for the suppression of acute liver injury and assess the underlying molecular mechanisms. At first, liver damage was examined by serum levels of AST, ALT, and AKP in WT or RIPK1-KO mice. As shown in Fig. 2A, serum AST, ALT, and AKP levels were markedly higher in WT-mice with LM infection than in a control group of uninfected WT mice. However, these effects were significantly reduced in mice with RIPK1 knockout or JQ1 pre-treatment. Moreover, to confirm whether JQ1 could effectively diminish the level of RIPK1-associated necroptosis, adenoviral vectors (Ad) over-expressing RIPK1 (AdRIPK1) and the negative control vectors (AdCtrl) were injected into the JQ1-treated mice 24 h before LM-induced acute liver injury. Administration of AdRIPK1 negated the protective role of JQ1 in reducing AST, ALT, and AKP levels. H&E and TUNEL staining showed that, after LM infection, WT-mice exhibited obvious necroptosis and histological changes in hepatic tissues, with significant up-regulation of cell death. These phenomena were alleviated to a great extent by RIPK1 knockout and JQ1 pre-treatment individually. In comparison, over-expressing RIPK1 abrogated the protective effects of JQ1 on histological alterations and cell death (Fig. 2B and C). There was considerably more RIPK1, RIPK3, p-MLKL, and BRD4 expression in groups with LM infection than in the WT/Ctrl group. JQ1 pre-treatment and RIPK1 deficiency both rendered the augmentation of RIPK1, RIPK3, pMLKL, and BRD4 less pronounced, demonstrating an inhibitory effect of JQ1 on RIPK1-associated necroptosis in acute liver injury by LM infection. However, promoting RIPK1 expression significantly abrogated the role of JQ1 in the repression of these signals (Fig. 2D–H). These findings above suggested that suppressing BRD4 expression by JQ1 could attenuate acute liver injury through repressing RIPK1 expression.
2.9. Immunohistochemical(IHC) analysis Hepatic tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections (5-μm thickness) were affixed to slides, deparaffinized, and then were subjected to hematoxylin and eosin (H&E) staining. For IHC staining, hepatic sections were incubated with primary antibody F4/80 (1:150 dilutions, Abcam, USA) overnight at 4 °C. After washing, the sections were incubated with HRP-conjugated secondary antibody (Beyotime), followed by 3,3′-diaminobenzidine (DAB) (Sigma Aldrich, USA) incubation. Then, the representative images were obtained using a light microscope. As for apoptosis in liver, the paraffin-embedded hepatic sections were stained by TUNEL assay (KeyGen Biotech, Nanjing, China) to calculate in vivo cell death according to the manufacturer’s protocols. TUNEL-stained liver tissues were visualized under a light microscope. 2.10. Mitochondrial membrane potential and mitochondrial permeability transition pore (mPTP) calculation The mitochondrial membrane potential was determined using the JC-1 probe (Beyotime) according to the manufacturer’s protocols. mPTP opening was determined as previously indicated using tetramethylrhodamine ethyl ester. The relative mPTP opening was calculated as a ratio to that of the control group [19]. 2.11. Mitochondrial DNA calculation Mitochondrial DNA was quantified according to previous study [20]. 2.12. Statistical analysis All results are presented as the mean ± standard error of the mean (SEM.). Statistical comparisons were performed using one-way analysis of variance (ANOVA), difference between two groups was analyzed using Student’s two-tailed t-test. P value < 0.05 for the difference was considered statistically significant. Details for materials and methods used in the study were exhibited in Supplementary part.
3.3. JQ1 alleviates hepatic inflammation in LM-infected mice Inflammation plays a critical role in regulating acute liver injury, which is associated with necroptosis [21]. Here, we established that pro-inflammatory cytokines and chemokines, including TNF-α, IL-6, IL1β, IL-18, MCP-1, and MCP-2, were highly induced by LM in liver tissue samples; however, the elevation of these factors triggered by LM was markedly abrogated by JQ-1 pre-treatment and by RIPK1 deletion. RIPK1 over-expression abolished the anti-inflammatory effects of JQ1, contributing to the severity of liver injury (Fig. 3A and B). In addition, hepatic p-IκBα and p-NF-κB expression levels stimulated by LM were greatly reduced by JQ-1 administration and RIPK1 deletion. In comparison, RIPK1 over-expression markedly abrogated the role of JQ1 in blocking IκBα/NF-κB signaling (Fig. 3C and D). Moreover, JQ1 pretreatment and RIPK1 knockout significantly reduced inflammatory cell recruitment in liver tissues from LM-infected mice, as evidenced by the down-regulated levels of expression of CD68. However, these effects regulated by JQ1 were markedly abrogated by RIPK1 over-expression (Fig. 3E). These results established that JQ1-suppressed inflammation in acute liver injury by LM infection took place mainly in an RIPK1dependent manner.
3. Results 3.1. BRD4/RIPK1 activation is associated with the progression of acute liver injury To calculate the effects of BRD4 on acute liver injury, changes in its expression were detected in vitro and in vivo. As shown in Fig. 1A and B, LLO-infected KCs showed significantly up-regulated expression of BRD4 from mRNA and protein levels compared to the Ctrl group. Meanwhile, the levels of expression of necroptosis markers, such as RIPK1, RIPK3, and p-MLKL, were found to be markedly induced by LLO stimulation in a dose-dependent manner (Fig. 1C). In vivo, we also found that levels of BRD4 expression in hepatic samples were markedly more induced by LM infection than in the control group (Fig. 1D and E). LM-infected mice also showed significantly increased expression of RIPK1, RIPK3, and p-MLKL in liver tissues (Fig. 1F). TUNEL staining showed that LLO treatment led to detectable cell death in KCs (Fig. 1G). Consistently, significantly increased numbers of TUNEL-positive cells in hepatic
3.4. JQ1 improves mitochondrial function by suppressing RIPK1 signaling The condition of mitochondria plays a critical role in meditating 3
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Fig. 1. LM infection up-regulates BRD4 expression in hepatic tissues of mice. (A) RT-qPCR and (B) western blot analysis for BRD4 in KCs treated with LLO (62.5, 125 or 250 ng/ml) for 8 h. (C) Western blot results for RIPK1, RIPK3 and p-MLKL in KCs incubated with LLO (62.5, 125 or 250 ng/ml) for 8 h.(D) RT-qPCR and (E) western blot analysis for BRD4 in liver tissues of LM-infected mice for the indicated time (0, 4, 8, 12, 16 or 24 h). (F) Western blot results for RIPK1, RIPK3 and pMLKL in hepatic samples of LM-infected mice for the shown time points (0, 4, 8, 12, 16 or 24 h). (G) KCs were exposed to LLO (62.5, 125 and 250 ng/ml) for 8 h, followed by apoptosis measurement using TUNEL staining. (H) TUNEL staining of liver sections from LM-infected mice at the indicated time. Data were expressed by means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001vs the Con group.
opposite results were detected in the change of mPTP opening in KCs as shown in Fig. 4G. Then, JC-1 staining further established that LLO-incubated KCs had significantly impaired mitochondrial membrane potential, which was rescued by JQ1 incubation or RIPK1 deletion. The protective role of JQ1 in improving mitochondrial membrane potential took place in an RIPK3-dependent manner, and similar results were detected in the mtDNA levels (Fig. 4J and K). Therefore, RIPK1 suppression was also required for improving mitochondrial function exerted by JQ1.
RIPK1-regulated necroptosis [22]. To further investigate the molecular mechanism underlying JQ1-modulated RIPK1 inactivation and suppression of necroptosis in LM-induced acute liver injury, mitochondrial function was evaluated in vivo and in vitro. As shown in Fig. 4A, TEM suggested that LM infection caused mitochondrial impairments in liver sections. JQ1 administration-improved mitochondrial was, however, abolished by RIPK1 over-expression in liver of LM-infected mice (Fig. 4A). We then found that hepatic ATP contents and mitochondrial membrane potential were significantly reduced by LM infection, and these effects were rescued by JQ1 administration or RIPK1 knockout. Over-expression of RIPK1 markedly abrogated the protective effects of JQ1 against mitochondrial function (Fig. 4B and C). Then, Western blot analysis was used to calculate the protein expression levels of signals associated with mitochondrial function, including DRP1, MFF, FIS1, PGAM5, and CypD [23,24]. LM-infected WT mice showed significantly higher levels of expression of DRP1, MFF, FIS1, PGAM1, and CypDin liver tissues than in the control, and the promotion of these proteins was attenuated by JQ1 and RIPK1 deletion. The inhibitory role of JQ1 in suppressing these molecules was found to operate in a RIPK1-dependent manner (Fig. 4D). To confirm the effects of JQ1 against mitochondrial dysfunction, the in vitro study was then performed using KCs isolated from the WT or RIPK1-KO mice. At first, MTT results suggested that the cell viability was visibly reduced by LLO stimulation, but was restored near to normal levels by JQ1 incubation or RIPK1 knockout. However, RIPK1 over-expression through AdRIPK1 transfection resulted in the reduction of cell viability (Fig. 4E). Consistently, ATP contents, Complex I activity, and Complex IV activity were found to be lower in LLO-incubated KCs, and it returned to near-normal levels due to JQ1 pre-treatment or RIPK1 deletion. RIPK1 over-expression markedly abolished the effects of JQ1 on the improvement of ATP, Complex I, and Complex IV after LLO stimulation (Fig. 4F, H and I). The
4. Discussion In this study, we found levels of BRD4 expression to be markedly upregulated in mice with acute liver injury induced by LM. The RIPK1/ RIPK3/MLKL signaling pathway was also activated after LM infection, contributing to the necroptosis and subsequent hepatic damage. RIPK1 genetic deletion attenuated necroptosis, inflammation, and mitochondrial dysfunction in hepatic tissues from LM-infected mice. Notably, suppressing BRD4 with JQ1 markedly reduced RIPK1 activation, inactivated RIPK3/MLKL signaling, repressed inflammatory response, and alleviated mitochondrial disturbance, which demonstrated the protective effects of JQ1 against acute liver injury associated with septicemia. Increasing RIPK1 expression markedly abrogated the protective effects of JQ1, indicating that JQ1-improved acute liver injury was largely RIPK1 dependent. To our knowledge, this is the first evidence that the effects of JQ1 on LM-induced necroptosis, which was focused on RIPK1 signaling. BRDs are crucial epigenetic “reader” proteins, which specifically recognize acetylated-lysine residues in nucleosomal histones, thus facilitating the recruitment of transcriptional proteins to chromatin [13,14]. The BRD4 inhibitor JQ1 was found to have anti-inflammatory, 4
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Fig. 2. JQ1 improves acute liver injury through suppressing RIPK1 activation after LM infection for 24 h. (A) Serum ALT, AST and AKP levels were assessed. (B) Up panel, H&E staining of hepatic sections; down panel, TUNEL staining of liver specimens. (C) Quantification of TUNEL-positive levels in hepatic samples. (D–H) RIPK1, RIPK3, p-MLKL and BRD4 protein levels in liver tissue were measured by western blotting analysis. Data were expressed by means ± SEM. **p < 0.01 and ***p < 0.001vs the Ctrl/WT group; +p < 0.05, ++p < 0.01 and +++p < 0.001vs the LM/WT group; #p < 0.05 and ##p < 0.01vs the LM/JQ1+AdCtrl group.
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Fig. 3. JQ1 alleviates hepatic inflammation in24 h of LM-infected mice. (A) ELISA analysis for TNF-α, IL-6, IL-1β, IL-18 and MCP-1 in liver samples. (B) RT-qPCR analysis for TNF-α, IL-6, IL-1β, IL-18, MCP-1 and MCP-2 in hepatic tissues. (C and D) Western blot analysis was used to determine p-IκBα and p-NF-κB protein expression levels in liver samples. (E)IHC staining for F4/80 in hepatic sections. Data were expressed by means ± SEM.*p < 0.05, **p < 0.01 and ***p < 0.001vs the Ctrl/WT group; +p < 0.05 and++p < 0.01vs the LM/WT group; #p < 0.05 and ##p < 0.01vs the LM/JQ1+AdCtrl group.
which was similar to the inhibitory role of RIPK1 knockout. Notably, over-expression of RIPK1 abrogated the anti-inflammatory effects of JQ1 in LM-challenged mice. These findings indicated that the JQ1suppressed inflammatory response was largely RIPK1-dependent. MLKL could lead to mitochondria fission through PGAM5 and DRP1 [33]. PGAM5 plays a significant role in regulating mitochondrial homeostasis and various necrotic death pathways [34]. The downstreaming executive events of necroptosis are RIPK3-induced activation of PGAM5 and mPTP opening [35]. Functional analysis clearly indicated that the activation of RIPK3 increased PGAM5 expression, which subsequently enhanced CypD expression and caused cells to undergo necroptosis by promoting the opening of mPTP [24]. Mitochondrial dysfunction has been linked to various hepatic diseases, including acute liver injury induced by LM and lipopolysaccharide [36]. PGAM5 can regulate mitochondrial fission to control mitochondrial dynamics [37]. DRP1, a large GTPase, plays an essential role in meditating the mitochondrial fission in mammalian cells [38]. The RIPK1/RIPK3 complex can initiate mitochondrial fission, which is involved in the promotion of inflammation [39]. PGAM5 is strictly necessary for concanavalinA (ConA)-induced hepatic necroptosis and liver injury [40]. In our study, we found for the first time that JQ1 treatment markedly alleviated mitochondrial impairment and dysfunction in livers of LM-infected mice and in LLO-exposed KCs, as proved by the reduced levels of expression of PGAM5, CypD, DRP1, MFF, and FIS1. MFF and FIS1 are hallmarks of mitochondrial fission, playing an essential role in inducing the disorder of mitochondria [23]. However, over-expression of RIPK3 was found to markedly abrogate the role of JQ1 in improving mitochondrial function, further indicating that JQ1-
anti-fibrotic, and anti-tumor effects [14,25,26]. More recently, changes in BRD4 expression were reported to be associated with necroptosis by regulating MLKL activation [14]. Necroptosis, a form of cell death, critically depends on RIPK3/MLKL signaling activation regulated by RIPK1 [11,12]. Acute liver injury is a life-threatening syndrome due because of its massive inflammation and cell death. Suppressing necroptosis is an effective means of treating acute liver failure as induced by d-galactosamine or lipopolysaccharide [27]. In this study, we further confirmed the effects of BRD4 on the necroptosis progression in mice with acute liver injury driven by LM. First, JQ1 treatment alleviated RIPK3 and MLKL activation in the livers of LM-infected mice, which was similar to the effects of RIPK1 knockout. The programmed necroptosis regulated by RIPK3 has served as an inflammatory form of cell death with critical functions in pathogeninduced and sterile inflammation [28]. RIPK3-regulated necrotic cell death promotes systematic inflammation and mortality [11]. Fatal septicemia is closely associated with systemic inflammatory response syndrome. This process is attributed to the excessive release of proinflammatory cytokines (TNF-α, IL-6, IL-18, and IL-1β) and chemokines (MCP-1 and MCP-2) [29,30]. In our study, we confirmed that LM infection resulted in inflammatory response in hepatic tissues, as evidenced by the significantly increased expression of TNF-α, IL-6, IL-18, IL-1β, MCP-1, and MCP-2 by activating NF-κB pathway. NF-κB is a pivotal signaling factor involved in inducing the secretion of pro-inflammatory factors [31]. BRD4 suppression by JQ1 is also effective for the blockage of NF-κB pathway, subsequently reducing the production of inflammatory molecules [32]. Consistently, JQ1 treatment markedly abrogated LM-induced inflammation by repressing NF-κB activation,
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Fig. 4. JQ1 improves mitochondrial function by suppressing RIPK1 signaling after LM infection for 24 h. (A) TEM for liver samples. The red asterisk referred the impaired mitochondrial. (B) ATP contents in hepatic tissues. (C) Mitochondrial membrane potential(Ψm) was measured. (D) Western blot analysis of signals associated with mitochondrial function, including DRP1, MFF, FIS1, PGAM5 and CypD in liver tissues. (E–K) KCs isolated from the indicated groups of mice were pretreated with JQ1 for 24 h, and then transfected with AdRIPK1 for another 24 h, followed by exposure to LLO (250 ng/ml) for final 8 h. Then, all cells were harvested for the following studies. (E) Cell viability was measured using MTT analysis. (F) ATP contents in KCs were tested. (G) Evaluation of mPTP opening. (H) Complex I activity and (I) Complex IV activity in cells were assessed. (J) Mitochondrial membrane potential (Ψm) was measured using JC-1 staining. (K) Calculation of cellular mtDNA levels.Data were expressed by means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001vs the Ctrl/WT group; +p < 0.05 and++p < 0.01vs the LM/WT or LLO/WT group; #p < 0.05 and ##p < 0.01vs the LM/JQ1+AdCtrlor LLO/JQ1+AdCtrl group.
Conflict of interest
alleviated acute hepatic injury was RIPK3-dependent. In conclusion, the results in this study highlighted the critical role of necroptosis in acute liver injury as regulated by the RIPK1/BRD4 signaling pathway. These findings established that BRD4 inhibition by JQ1 could ameliorate necroptosis, inflammation, and mitochondrial dysfunction mainly through RIPK1 inhibition, consequently repressing septicemia-associated acute hepatic injury. BRD4 inhibitor might be an effective approach for the treatment of acute hepatic disease. Nevertheless, understanding of JQ1-induced RIPK1 inactivation is still poor. Further study is required to render our understanding more comprehensive.
The authors see no any conflict of interest in this study. Acknowledgments We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2020.109818. 7
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