Bromodomain protein 4 inhibitor JQ-1 alleviates hepatic ischemia-reperfusion injury by blocking the NLRP3/caspase-1 pathway

Abstract

BACKGROUND:

Hepatic ischemia-reperfusion (I/R) injury is a major challenge in liver surgery and transplantation. Bromodomain protein 4 (BRD4) has emerged as a promising target due to its role in oxidative stress and inflammation. JQ-1, a specific BRD4 inhibitor, has shown protective effects on organs suffering I/R injury. This study aims to investigate the expression of BRD4 in liver tissues after I/R injury and to explore its role in this process using JQ-1 both in vivo and in vitro.

METHODS:

Our study established a mouse model of hepatic I/R injury and investigated the protective effect of JQ-1. We compared the histological features, BRD4 expression, and liver enzyme levels between JQ-1-treated and untreated groups. Additionally, the antioxidant properties of JQ-1 were analyzed in RAW 264.7 cells by evaluating cytokine expression, NLRP3 inflammasome activity, and reactive oxygen species production.

RESULTS:

BRD4 was abundantly expressed in liver tissues after hepatic I/R injury, while JQ-1 treatment had antioxidant and hepatoprotective effects. JQ-1 also suppressed pro-inflammatory cytokine release in vitro. Furthermore, we clarified the mechanism by which JQ-1 enhances liver injury recovery through Kupffer cells by blocking the NOD-like receptor thermal protein domain-associated protein 3 (NLRP3)/caspase-1 pathway.

CONCLUSION:

JQ-1 has potential as a pre-clinical emergency therapy for hepatic I/R injury. Its ability to inhibit BRD4 and modulate the inflammatory response in Kupffer cells offers a promising avenue for future clinical intervention.

INTRODUCTION

Hepatic ischemia-reperfusion (I/R) injury, which occurs during liver resection or transplantation, remains a substantial challenge for the postoperative survival and quality of life of recipients.^[1-3] Various factors, including the upregulation of pro-inflammatory cytokines and the activation of inflammasomes, reportedly contribute to I/R.^[4,5] Additionally, accumulating evidence points to reactive oxygen species (ROS) as a primary contributor to hepatic I/R injury.^[4,6] This process is characterized by a significant reduction of glutathione and limited formation of protein adducts triggered by reactive N-acetyl-4-benzoquinone imine, resulting in amplified oxidant stress, particularly in mitochondria, ultimately leading to I/R.^[7,8] Furthermore, sterile inflammation, involving the recruitment of neutrophils and monocyte-derived macrophages to the liver, is associated with I/R.^[9,10] Despite numerous studies exploring novel strategies to alleviate hepatic I/R injury,^[11,12] their effects on postoperative survival and recipient quality of life remain uncertain.^[13,14]

The bromodomain and extra-terminal (BET) family consists of proteins that specifically identify acetylated lysine residues, consequently regulating gene transcription and contributing to the initiation and progression of various inflammatory diseases. Bromodomain protein 4 (BRD4), a member of the BET family, is associated with inflammation-induced injury involving ROS synthesis and cytokine processes. BRD4 inhibitors have been used to alleviate inflammation and reduce the release of pro-inflammatory cytokines and ROS.^[4] The BRD4 inhibitor JQ-1 has showed efficacy in the treatment of renal or myocardial I/R.^[15,16] However, the effect of JQ-1 on hepatic I/R injury remains unclear.

In this study, we aim to investigate the expression of BRD4 in liver tissues after I/R injury and to uncover its role in this process using JQ-1, a specific BRD4 inhibitor, both in vivo and in vitro. Furthermore, we aim to explore the related mechanism and pathways.

METHODS

Animals

Male wild-type C57BL/6 mice, aged 6-8 weeks, were acquired from the Shanghai Experimental Animal Center. The mice were housed in a specific pathogen-free (SPF) environment. All animal procedures were ethically approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medicine University (IACUC-20230717-11).

The mice were randomly divided into three groups: sham, I/R, and JQ-1 groups, with five mice in each group. After an overnight fasting period, mice were anesthetized with pentobarbital (100 mg/kg). Following laparotomy, microartery clamps were used to occlude the branch of the hepatic artery, the median, and the left hepatic portal vein. Blood supply to the caudal and right lobes was preserved. After 90 min of ischemia, the blood supply to the ischemic liver lobes was restored by removing the clamps. The abdomen was then closed. After 6 h of reperfusion, mice were reanesthetized and euthanized by cervical dislocation. Serum samples were collected, and the left lobes of the liver were obtained for histological analyses. The sham group underwent the same surgical procedure without hepatic portal occlusion. In the JQ-1 group, JQ-1 (50 mg/kg) was intraperitoneally injected 2 h before the surgical procedure, following our previous research protocols.^[17,18] The same dose of phosphate buffered saline (PBS) was injected as a control in the I/R group.

Histological, immunohistochemical and immunofluorescence analyses

Liver samples were excised, fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. The embedded samples were then cut into 4 μm-thick slices. For histological staining, tissue sections were deparaffinization and stained with hematoxylin and eosin (H&E). The images of stained sections were captured using optical microscopy. The Suzuki Score was used to assess the extent of liver tissue damage.^[19] Immunohistochemical (IHC) analyses were conducted using standard procedures. In brief, the samples were incubated with the anti-BRD4 antibody (ab128874, Abcam, USA) overnight at 4 °C, followed by staining with a secondary antibody at 37 °C for 1 h. For immunofluorescence analysis, deparaffinized sections were incubated with F4/80 (GB113373, Servicebio, China), NLRP3 (GB114320, Servicebio, China), or Caspase-1 (GB11383, Servicebio, China) according to the manufacturer’s instructions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay was performed using the TUNEL assay kit (C1086, Beyotime, China) following the manufacturer’s instructions. The apoptotic ratio of hepatocytes was visualized using a fluorescence microscope.

Cells and reagents

The murine macrophage cell line RAW 264.7 (CL-0190) was purchased from Wuhan Pricella Life Technology Co., Ltd (China). JQ-1 (S7110) was obtained from Selleck Chemicals (USA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Biological Industries (Israel). H₂O₂ was purchased from Sigma-Aldrich (USA).

Cell proliferation assay

RAW 264.7 cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin. Cells, seeded in 96-well plates, were treated with various concentrations of JQ-1 (10 nmol/L, 50 nmol/L, 100 nmol/L, 500 nmol/L, 1 μmol/L, and 2 μmol/L) for 24 h. At the end of the incubation period, the previous medium was replaced with a fresh medium containing 10% Cell Counting Kit-8 solution (C0037, Beyotime, China). After 2 h of incubation, the absorbance at 450 nm was measured using a microplate reader (Multiskan FC, Thermo Scientific, USA). Then, cell proliferation was calculated according to the manufacturer’s protocol.

Serum liver enzyme assay and enzyme-linked immunosorbent assay (ELISA)

For the three groups of mice, the serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using an automatic biochemical analyzer (Model 7600 Series Automatic Analyzer, Hitachi, Japan).

RAW 264.7 cells were seeded in 12-well plates and exposed to 300 μmol/L H₂O₂ for 24 h, with or without treatment with JQ-1 (500 nmol/L). Then, the supernatant medium was collected, and the detached cells were centrifuged. Following centrifugation, the cytokine secretion (interleukin [IL]-10, IL-1β, IL-6, and IL-18) of RAW 264.7 cells was measured using ELISA kits, following the manufacturer’s protocol.

ROS analysis

RAW 264.7 cells were seeded in 12-well plates and exposed to 300 μmol/L H₂O₂ for 24 h, with or without treatment with JQ-1 (500 nmol/L). Subsequently, the centrifuged cells were collected, and the DCFH-DA probe was loaded. After the incubation and washing steps, the level of ROS in RAW 264.7 cells was measured using an ROS assay kit (S0033, Beyotime, China) following the manufacturer’s instructions.

Western blot analysis

Protein extraction from RAW 264.7 cell samples was carried out, and the following antibodies were utilized for detection: NLRP3 (#15101, CST, USA), thioredoxin-interacting protein (TXNIP) (#14715, CST, USA), Caspase-1 (#83383, CST, USA), and β-actin (#4970, CST, USA). The Western blot analysis was conducted following standardized procedures.

Statistical analysis

All quantitative data are presented as the means ± standard deviation (SD). The two-tailed independent student’s t-test was employed to assess the differences between the groups. For continuous data with a normal distribution, one-way ANOVA was utilized for comparisons among multiple groups. If significant differences were observed, the SNK method was applied for further analysis. For the statistical analysis of non-normally distributed data, the Kolmogorov-Smirnov test was employed to assess normality. Non-normally distributed data were represented using interquartile ranges, and two-sample rank sum tests were conducted. The results were considered statistically significant at a P-value < 0.05.

RESULTS

The expression of BRD4 and the serum levels of ALT and AST during hepatic I/R injury

Considering the fascinating effects of BRD4 in resisting oxidative stress and inflammation, which are important factors contributing to hepatic I/R injury, we embarked on investigating the expression of BRD4 in the liver tissues of hepatic I/R injury mice. The serum levels of ALT and AST were significantly increased after the establishment of the I/ R model, as shown in Figure 1A and 1B. Immunohistichemical staining revealed that BRD4 was barely expressed in normal liver tissues, while its level was elevated in hepatic I/R injury mice (Figures 1C and 1D). These findings suggest a potential involvement of BRD4 in hepatic I/R injury.

Figure 1. The expression of BRD4 and the serum levels of ALT and AST during hepatic I/R injury. A: Representative immunohistochemical staining of BRD4 in liver tissues. Magnification, 100×; scale bar, 100 μm. B: Quantification of immunohistochemical staining of BRD4 in liver tissues of mice from the sham and I/R groups. C, D: The concentration of serum ALT and AST in the sham group and the I/R group. BRD4: bromodomain protein 4; I/R: ischemia/reperfusion; ALT: alanine aminotransferase; AST: aspartate aminotransferase. ***P<0.001.

JQ-1 protects against hepatic I/R injury by inhibiting the expression of BRD4

Due to the increased expression of BRD4 in liver tissues after I/R injury, we proceeded to explore whether JQ-1, a specific BRD4 inhibitor, could ameliorate hepatic I/R injury in mcie. As shown in Figure 2A, in contrast to the extensive edema and central venous congestion observed in the liver tissue of the I/R group, after 24 h of treatment, the liver cells in the JQ-1 group displayed normal morphology, no significant cell edema, and no notable inflammatory cell infiltration or tissue congestion in the liver parenchyma. Consistent with the Suzuki score results (Figure 2B), the expression of BRD4 was lower in the JQ-1 group than that in the I/R group (Figure 2C). Furthermore, the serum ALT and AST concentrations were elevated in the I/R group and downregulated in the JQ-1 group. However, the serum concentrations of ALT and AST in the JQ-1 group were higher than those in the sham group (Figures 2D and E). In summary, JQ-1 treatment improves liver function, indicating its hepatoprotective efficacy in vivo.

Figure 2. Inhibition of BRD4 by JQ-1 protects against hepatic I/R injury. A: H&E staining and BRD4 immunohistochemical staining in liver tissues from the sham, I/R, and JQ-1 (50 mg/kg, i.p.) groups. Magnification, 100×; scale bar, 100 μm. B, C: Suzuki score and BRD4 relative expression of the sham, I/R, and JQ-1 groups of mice. D, E: The concentration of ALT and AST of the three groups. BRD4: bromodomain protein 4; I/R: ischemia/reperfusion; ALT: alanine aminotransferase; AST: aspartate aminotransferase. ***P<0.001, **P<0.01, *P<0.05.

The cytoprotective effect of JQ-1 in vivo

Inflammation and oxidative stress are the primary factors to induce cell damage after I/R injury. Therefore, we assessed the protective effect of JQ-1 on I/R-induced cell apoptosis in vivo via immunofluorescence staining. After I/R injury, an increase in the proportion of TUNEL-positive cells was observed in the I/R group compared to the sham group (Figure 3). The administration of JQ-1 (50 mg/kg) resulted in a significant reduction in the percentage of TUNEL-positive cells in liver tissues. These results suggest that JQ-1 might exert cytoprotective effects and ameliorate hepatic I/R injury.

Figure 3. The cytoprotection activity of JQ-1 in vivo. Representative immunofluorescence staining of TUNEL (green) and DAPI (blue) in liver tissues of the sham, I/R and JQ-1 (50 mg/kg) groups. Magnification, 100×; scale bar, 100 μm. I/R: ischemia/reperfusion. **P<0.01.

JQ-1 reduces the levels of inflammatory cytokines and ROS in vitro

We then embarked to clarifying the potential mechanism underlying the hepatic protective effect mediated by JQ-1. A close relationship between BRD4 and macrophages has been reported in previous studies.^[20-22] Due to technical limitations, we used the murine macrophage cell line RAW 264.7 to mimic the macrophage response in vitro. As illustrated in Figure 4A, 1 µmol/L of JQ-1 significantly inhibited RAW264.7 cell proliferation. To avoid compromising cell survival, we selected a concentration of 500 nmol/L for subsequent H₂O₂-induced oxidative damage experiments for RAW264.7 cells. Compared with the H₂O₂ group, the release of the anti-inflammatory cytokine IL-10 in H₂O₂+JQ-1 group was elevated, while the production of pro-inflammatory cytokines IL-1β, IL-6, and IL-18 decreased (Figures 4B and C). Additionally, we assessed the ability of JQ-1 in inhibiting ROS generation. After stimulation with H₂O₂ at 300 µmol/L for 24 h, an increase of ROS levels was observed in the H₂O₂ group. The supplementation of JQ-1 resulted in a significant decrease in ROS levels (Figure 4D). These findings indicate that JQ-1 exhibits significant antioxidant effects to defend oxidative stress.

Figure 4. JQ-1 reduced the levels of pro-inflammatory cytokines and ROS in vitro. A: Cell proliferation ratio of RAW264.7 cells treated with JQ-1 at various concentrations for 24 h. B: The expression of IL-10 in the control, H₂O₂-induced, and H₂O₂+JQ-1 (500 nmol/L) groups for 24 h. C: The expression of IL-1β, IL-6 and IL-18 in the control, H₂O₂-induced, and H₂O₂+JQ-1 (500 nmol/L) groups for 24 h. D: The ROS level in RAW264.7 cells in the control, H₂O₂-induced, and H₂O₂+JQ-1 (500 nmol/L) groups for 24 h. ROS: reactive oxygen species; IL: interleukin; ***P<0.001, **P<0.01, *P<0.05.

JQ-1 alleviates the hepatic I/R injury by inhibiting the aggregation of NLRP3/caspase-1-positive macrophages

Considering the crucial role of macrophages in hepatic I/R injury, we explored the molecular mechanisms underlying macrophage activation. Given the significance of NLRP3 inflammasome activation in macrophage under I/R injury and the ability of JQ-1 in inhibiting NLRP3 inflammasome activation as reported previously,^[20,23,24] we hypothesized that the therapeutic effect of JQ-1 on hepatic I/R injury might result from the suppression of the NLRP3/caspase-1 pathway. As depicted in Figure 5A, immunofluorescence staining suggested that NLRP3-positive macrophages aggregated in liver tissues subjected to I/R injury, while JQ-1 treatment mitigated the infiltration of these macrophages. Similarly, caspase-1 positive macrophages were diminished after JQ-1 treatment in hepatic I/R injury mice (Figure 5B). These findings indicate that the aggregation of NLRP3/caspase-1-positive macrophages might contribute to hepatic I/R injury, and JQ-1 significantly attenuated these reactions.

Figure 5. JQ-1 alleviate hepatic I/R injury by inhibiting the aggregation of NLRP3/caspase-1-positive macrophages. (A) Representative immunofluorescence staining of F4/80 (green), NLRP3 (red), and DAPI (blue) in liver tissues of the sham, I/R and JQ-1 (50 mg/kg) groups. Magnification, 100×; scale bar, 100 μm. (B) Representative immunofluorescence staining of F4/80 (green), Caspase-1 (red), and DAPI (blue) in liver tissues of the sham, I/R and JQ-1 (50 mg/kg) groups. Magnification, 100×; scale bar, 100 μm. ***P<0.001, **P<0.01, *P<0.05.

JQ-1 inhibits NLRP3/caspase-1 pathway in macrophages induced by H₂O₂

The protein expression of NLRP3/caspase-1 was further investigated by western blot in vitro. Compared to that in the H₂O₂-treated group, the treatment with JQ-1 reduced the protein level of NLRP3 in RAW264.7 cells (Figure 6). The trends of expression levels of TXNIP and Cle-Caspase-1 were also consistent with NIRP3. These results indicate that JQ-1, a selective BRD4 inhibitor, could enhance antioxidant and cell-protective effects by blocking the NLRP3/caspase-1 pathway.

Figure 6. JQ-1 alleviates hepatic I/R injury by blocking the NLRP3/caspase-1 pathway. The protein levels of TXNIP, NLRP3, and Cle-Caspase-1 of RAW264.7 cells in the control, H₂O₂-induced (300 μmol/L), and H₂O₂+JQ-1 (500 nmol/L) groups for 24 h. **P<0.01, *P<0.05.

DISCUSSION

Hepatic I/R injury frequently occur in various clinical emergencies, including liver transplantation, liver resection surgery, abdominal hemorrhage, trauma, and shock.^[25] This may lead to liver dysfunction, significantly affecting patient outcomes.^[26,27] Although multiple factors have been implicated in the process of hepatic I/R injury, the underlying mechanisms are not fully elucidated.^[28] BRD4, a member of the bromodomain and extraterminal domain family, is known to identify and bind to multiple acetylation sites on acetylated histones.^[29] Several studies have indicated the involvement of BRD4 in inflammation and oxidative stress damage.^[30,31] Our previous research demonstrated that BRD4 inhibition could prolong the survival of heart grafts by enhancing myocardial autophagy.^[32] Additionally, BRD4 inhibition has been reported to reduce cytokine secretion in macrophages.^[33,34] The small molecule compound JQ-1 is utilized to inhibit BRD4, contributing to the reduction of inflammation and acting as an antioxidant in the management of various inflammatory disorders.^[20,35,36] However, the specific effect of JQ-1 on hepatic I/R injury remains unclear.

In this study, we observed an increase in BRD4 levels following hepatic I/R injury in vivo. JQ-1 treatment mitigated hepatic congestion and reduced inflammatory cell infiltration in liver tissues. Immunofluorescence tests revealed that JQ-1 affected mice liver tissues, producing anti-inflammatory effects. In the cell experiments, RAW 264.7 cells were used to mimic Kupffer cells. Treatment with JQ-1 was indicated to protect RAW264.7 cells against oxidative damage induced by H₂O₂. Furthermore, we observed the involvement of the NLRP3 pathway in the mechanism of BRD4 inhibition by JQ-1. Considering that BRD4 plays a crucial role in transcriptional repression and NLRP3 inflammasome activity is essential to the inflammatory response,^[37] we identified a molecular connection between BRD4 and NLRP3 in hepatic I/R injury.

However, there are some limitations to this study. First, the exploration of the downstream pathway of JQ-1 did not involve the overexpression or knockdown of NLRP3. Second, cell experiments employing RAW264.7 cells may not fully capture the complexity of human immune responses due to the inherent differences between a murine macrophage cell line and primary human cells.^[38] While RAW264.7 cells are commonly used to mimic Kupffer cells in the liver and to screen for anti-inflammatory drugs, they still differ from the environment of macrophages in vivo, which may compromise the precision of our research. Third, although we observed the colocalization of NLRP3/caspase-1 and F4/80 fluorescence, the absence of Gdcl3 or liposomes to eliminate Kupffer cells constrained the further validation and application of our research.^[19,39]

CONCLUSION

Our findings suggest that BRD4 may serve as a therapeutic target for hepatic I/R injury. The inhibition of BRD4 by JQ-1 exhibits antioxidant and hepatocellular protective effects in vivo, along with a reduction in pro-inflammatory cytokine levels in vitro. JQ-1 may exert its effects by inhibiting the NLRP3/caspase-1 pathway in macrophages.

Funding: None.

Ethical approval: All animal studies were approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medicine University (IACUC-20230717-11).

Conflicts of interest: The authors have no conflict of interest.

Author contributions: WHG conceptualized the study and designed the methodology. LW conducted the experimental procedures and analysis. LW wrote the original draft of the manuscript. WHG reviewed and edited the manuscript. All authors approved the final version of the manuscript.