BRD4 inhibition by JQ1 protects against LPS-Induced Cardiac Dysfunction by inhibiting SIRT1-dependent activation of NLRP3 in�ammasomes

JQ1, a BRD4 protein inhibitor, �rst identi�ed because of its therapeutic role in cancer, has gradually demonstrated a protective effect on the heart in recent years; however, it is unclear whether JQ1 also plays a role in LPS-induced cardiac dysfunction. This paper aims to investigate the effects of the BRD4 inhibitor JQ1 on LPS-induced cardiac dysfunction and its mechanism. In the experiments, we found that BRD4 was signi�cantly upregulated in the hearts of LPS-treated mice.JQ1 treatment improved survival and cardiac function in LPS-treated mice and reduced cardiomyopathologic injury, in�ammation, and oxidative injury.JQ1 treatment similarly reduced the release of lactate dehydrogenase and in�ammatory factors in H9C2 cells treated with LPS.JQ1 signi�cantly upregulated silent information regulator 1 (SIRT1) expression and suppressed the upregulation of NOD-like receptor protein 3 (NLRP3), cleaved caspase-1, and GSDMD in heart tissues induced by LPS.Meanwhile, we obtained the same results in H9C2 cells treated with LPS. The administration of the SIRT1 inhibitor (EX527) intervention partially blocked the JQ1-mediated downregulation of NLRP3, cleaved caspase-1, GSDMD in LPS-induced H9C2 cells. Therefore, we propose that JQ1 can improve LPS-induced cardiac dysfunction by inhibiting SIRT1-dependent activation of NLRP3 in�ammasomes, which may be a promising strategy for treating sepsis cardiomyopathy.


Introduction
Sepsis is a complex multi-organ dysfunction syndrome Cardiac contraction and diastolic dysfunction are often seen in patients with severe sepsis [18].It should be noted that cardiovascular disease remains a signi cant cause of morbidity and mortality worldwide [6].Lipopolysaccharide(LPS), a signi cant component of the outer membrane of Gram-negative bacteria that can trigger pathologies such as systemic in ammatory response syndrome (SIRS) and septic shock, has been widely used in the establishment of models of cardiac dysfunction in sepsis [20].Therefore, it is crucial to explore the mechanisms of LPS-induced cardiac dysfunction.NOD-like receptor protein 3 (NLRP3) is an intracellular protein complex that regulates the body's in ammatory response [14].At the onset of in ammation, The NLRP3 is rapidly triggered for activation, activated NLRP3 binds to apoptosis-associated speck-like protein (ASC), then recruits caspase 1 to assemble into an in ammasome complex prompting the release of proin ammatory cytokines IL-1b and IL-18 and upregulating GSDMD, leading to pyroptosis [15,17,34].Previous studies have shown that NLRP3 in ammasome activation in cardiac broblasts during sepsis-induced maturation and release the in ammatory factors such as IL-1β, inhibiting activation of the NLRP3 in ammasome in myocardial broblasts, can alleviate LPS-induced myocardial dysfunction and improve survival in mice with peritonitis sepsis [40].Similarly, carbon monoxide molecules can also alleviate myocardial dysfunction in sepsis mice by inhibiting activation of NLRP3 in ammasome [39].Silent information regulator 1 (SIRT1) is a member of the Sirtuins family and is a conserved nicotinamide adenine dinucleotide (NAD+)dependent histone deacetylase.SIRT1 is primarily located in the mammalian nucleus and exerts its functions through acetylation activity.Recent studies have shown that SIRT1 can regulate NLRP3 to inhibit the onset of in ammation and delay the in ammatory response [13,16,24].Bromodomain-containing protein 4 (BRD4) belongs to the bromine and extra-terminal domain (BET) protein families.It binds to acetylated histones and transcription factors through its bromide domain to regulate various pathophysiological activities, including in ammation and cancer [25,29].JQ1 is one of the bromine domain inhibitors of BRD4 and has a therapeutic effect on various diseases [1,7,19].In recent years, there has been increasing evidence that JQ1 plays a role in cardiovascular disease.In cardiovascular animal models, JQ1 can reduce myocardial injury caused by ligation of the left coronary artery [32], reduce pulmonary hypertension, and prevent diabetic cardiomyopathy induced by a high-fat diet [22].Taken together, these data suggest that the BRD4 protein is involved in cardiovascular disease.
Recently, studies have shown that JQ1 is an effective regulator of SIRT1.In animal models, JQ1 can regulate autophagy, apoptosis, oxidative stress, and other cellular responses by upregulating SIRT1 [7,11,27,37].However, the role of SIRT1 in the protection of JQ1 against LPS-induced cardiac dysfunction is unclear.Therefore, this study investigates whether BRD4 protein inhibitor JQ1 regulates LPS-induced NLRP3 activation in mice by activating SIRT1, thus playing a protective role in against LPS-induced cardiac dysfunction.

Grouping and treatment of experimental animals
C57BL/6J male mice, aged 8-10 weeks(20-25 g), were purchased from the Institute of Experimental Animal Science, Hubei Medical University(Shiyan, China).Before the experiments, the animals were kept in a thermostatic environment with regular 12-hour light/dark cycles and provided with standard food and water.Forty-eight mice were randomly divided into 3 groups (6 mice in each group):(1) control group, (2) LPS group, (3) JQ1+ LPS group.Mice in LPS group were intraperitoneally injected with LPS 7.5mg/Kg(from Escherichia coli,serotype 0111: B4,Sigma,dissolved in 0.9% NaCl).Mice in the LPS+JQ1 group were given are protective treatment with 50mg/Kg JQ1(MedChem Express, HY-13030, and dissolved in 10% DMSO) intravenously 1 hour before LPS stimulation.The dose of JQ1 was based on a previous study [31].Mice in the control group were injected with DMSO in the caudal vein.Six mice in each group were euthanized after 12 h of LPS stimulation.Serum and heart tissue were collected and stored at −20℃for subsequent experiments(Fig.1).The survival rate of the remaining mice was recorded every 12 h until 72 h after LPS stimulation.

Echocardiography
Cardiac function was assessed by echocardiography after 12 hours of LPS stimulation.Mice were anaesthetized with sevo urane and xed in the supine position.Echocardiography was collected using a Mindray Resona7 imaging system equipped with a 20 MHz linear transducer.Left ventricular enddiastolic diameter (LVDD) and left ventricular end-systolic diameter (LVSD) of mice in each group were detected, and left ventricular ejection fraction (LVEF) and left ventricular short-axis shorting rate (LVFS) were calculated according to the built-in program.The mean values of the three measurements were recorded.

Measurement of Creatine Kinase-MB(CK-MB), Lactate Dehydrogenase (LDH), IL-6, IL-18
Serum and cell supernatants were collected and CK-MB, LDH, IL-1βand IL-18 levels in serum and cell supernatants according to instructions.The CK-MB and LDH kits were purchased from Nanjing Jiancheng Bioengineering Institute, China.The IL-1βELISA kit and the IL-18 ELISA kit were purchased from MULTISCIENCES (LIANKE) BIOTECH, CO., LTD

Oxidation index determination
The activity of superoxide dismutase (SOD), the activity of glutathione peroxidase (GSH-Px), and the content of maldialaldehyde (MDA) were determined using the corresponding kit (Nanjing Jianchen Bioengineering Institute, China).After various treatments of cardiomyocytes and cardiac tissue, the cells and tissue homogenates were collected and fragmented in 500µl PBS using an ultrasonic crush, followed by centrifugation at 1200 rpm at 4℃for 10 min.The supernatant was collected to determine SOD activity, GSH-PX activity, and MDA content using the corresponding kit according to the manufacturer's instructions.

Histological Staining
The rats were killed by neck removal, and the cardiac tissue was obtained.The cardiac tissue was xed with 4%(φ) paraformaldehyde, and then para n sections were prepared.Then HE staining was performed (baking slices -xylene dewaxing hydration -hematoxylin staining -acid ethanol solution differentiation -ethanol dehydration and transparency -neutral gum sealing slices).Finally, the changes in cardiac tissue were observed under a microscope.Three hearts were analyzed in each group.

CCK8 assay
H9C2 cells in the logarithmic growth stage and good growth state were inoculated into 96-well plates at a density of 3×104/ mL.JQ1 with nal concentrations of 0.25, 0.5, 1, 2, and 4µM were added into the cells.H9C2 cells supplemented with DMEM medium were set as the negative control group, and DMEM medium was set as the blank group.Each group has 3 duplicate holes.After 48h of culture, the absorbance (OD) of each well was measured at 450 nm using an automatic enzyme plate analyzer.The inhibitory rate of cell growth was calculated, and the inhibitory rate (%)=[1 −(OD value of the experimental group − OD value of the blank group)/(OD value of the control group − OD value of the blank group)]×100%.
2.9 Fluorescent staining of reactive oxygen species (ROS) H9C2 cells were seeded in 12-well plates, and the media was discarded after 12 h of drug intervention.DCFH-DA probes were diluted with serum-free medium with 4 ′,6-diamine-2-benzene-index (DAPI) blue in 1∶1500, and incubated in the incubator for 30 min.The medium was discarded, and the cells were washed twice with a serum-free medium.The staining level of cells was observed under uorescence microscope, and the uorescence luminosity value was detected.

Real-time PCR analysis
Total RNA was extracted from mouse myocardial tissue and H9C2 cells with Trizol reagent.cDNA was synthesized from total RNA using a reverse transcription kit.Ampli cation and quantitative RT-PCR analysis were performed using 7300 Real-Time PCR System (A p p l i e d B i o s y s t e m s) on a SYBR Green.The primer sequences used are as follows: 5′-CCATGGACATGAGCACAATC-3′(forward primer) and5′-TGGAGAACATCAATCGGACA-3′ (reverse primer) for the mouse BRD4 gene; 5′-CCGTGGCAAACTGGTACTTT-3′(forward primer) and5′-GACGCCAACATAGACCACCT-3′ (reverse primer) for the mouse SIRT1 gene; 5′-GACA TGCCGCCTGGAGAAAC -3′ (forward primer) and 5′-AGCCCAGGA TGCCCTTTAGT-3′ (reverse primer) for GAPDH (used as an internal reference control).The results were quanti ed using the 2-ΔΔCT method.

Statistical Analyses
All data in our study were expressed as mean ±SEM and statistically analyzed using GraphPad Prism software version 8.0.The comparison between the two groups was performed using the Two-tailed Student's t-test.Others used one-way analysis of variance (One-way ANOVA).P < 0.05 was considered statistically signi cant.

Cardiac BRD4 expression is upregulated in LPS -treated mice
To determine the relationship between BRD4 and LPS-induced cardiac dysfunction, mice were intraperitoneally injected with LPS (7.5 mg/kg) for 12 hours.Then we took mice cardiac tissue; the levels of BRD4 were detected by Rt-PCR and Western Blotting.Before euthanasia, mice injected with LPS developed typical signs of septic shock, such as decreased motor activity, clammy skin, anorexia, and lethargy.The results showed that the mRNA (Fig. 2a) and protein expression (Fig. 2b, c) of BRD4 were signi cantly increased in the hearts of LPS-stimulated mice compared with the control group.These ndings suggest that BRD4 protein may play a role in LPS-induced cardiac dysfunction.

BRD4 inhibitor JQ1 can improve the survival rate and cardiac dysfunction in LPS -treated mice
To test the effect of JQ1 on LPS-induced cardiac dysfunction in mice, we administered JQ1 through intravenous tail injection for pre protection for 1 hour, followed by intraperitoneal injection of LPS 7.5mg/Kg to induce myocardial injury.As shown in Figure 3A, the 72-h survival of LPS mice decreased to 41.6% compared to the control group.In contrast, treatment with 50mg /kg JQ1 signi cantly improved the survival rate to 75%.Echocardiography showed that LPS signi cantly reduced EF, FS, while treatment with JQ1 reversed these alterations (Fig. 3b, c, d).In addition, H&E staining of cardiac tissue revealed disrupted myocardial cell arrangement in the LPS group and increased cardiomyocytes compared to controls; in contrast, JQ1 treatment signi cantly alleviated these pathological abnormalities (Fig. 3b).LPS induced myocardial injury, as evidenced by signi cantly increased levels of LDH and CK-MB as compared to controls, and those of which were reduced by treatment with JQ1 (Fig. 3e, f).All these above data illustrate the protective effect of JQ1 on LPS-induced myocardial injury.

BRD4 inhibitor JQ1 reduces in ammation and oxidative damage in the heart tissue of LPS-treated mice
To assess the in ammatory response in mice, we performed immuno uorescence staining of in ltrating immune cells in myocardial tissue with a CD45 antibody and examined IL-1β, IL-18 levels in serum.The results showed that in myocardial tissue, the number of CD45-positive in ltrating immune cells in myocardium and serum IL-1β, IL-18 levels were signi cantly upregulated by LPS, and these effects were reversed by JQ1 (Fig. 4a, b, c).In addition, in assessing oxidative damage levels in heart tissue, we found that LPS treated mice had increased MDA content and signi cantly decreased SOD activity and GSH-PX activity, these alterations were similarly reversed by JQ1 treatment (Fig. 4d, e, f).These results demonstrate that JQ1 reduces in ammation and oxidative stress response in heart tissue of LPS treated mice.

BRD4 inhibitor JQ1 reduces LDH release, in ammation, and oxidative damage in LPS-treated H9C2 cells
To investigate the protective effect of JQ1 on LPS-treated H9C2 cells, cell damage indices, in ammatory factors, and oxidative damage indices were measured.In the CCK8 experiment, we found that JQ1 had no effect on cell viability at 0.25 and 0.5µM but decreased cell viability at 1µM (Fig. 5a).When the LDH level in the cell culture supernatant was measured, it was found that the LDH level decreased signi cantly when the JQ1 dose was 0.5µM (Fig. 5b).Therefore, 0.5µM JQ1 was selected as the optimal concentration for subsequent in vitro experiments.In addition, LPS treatment increased the release of IL-1βand IL-18 in H9C2 cells, whereas JQ1 treatment signi cantly reversed these LPS-induced effects (Fig. 5c, d).In the evaluation of oxidative damage indexes, compared with the control group, ROS uorescence was signi cantly enhanced after LPS induction, and SOD activity and GSH-Px activity were decreased.At the same time, JQ1 reversed these changes(Fig.5e, f, g).These results indicate that JQ1 has a protective effect on LPS-treated H9C2 cells.

JQ1 upregulates SIRT1 expression and inhibits NLRP3 activation in vivo and in vitro
To explore the protective mechanism JQ1 against LPS-induced myocardial injury, we detected the mRNA and protein expression levels of SIRT1, and it was found that LPS signi cantly reduced the mRNA and protein levels of SIRT1 in cardiac tissues, while JQ1 reversed these changes (Fig. 6a,b).At the same time, we detected the protein expression levels of NLRP3, Cleaved caspase-1, and GSDMD and found that LPS signi cantly upregulated the protein expression levels of NLRP3; Cleaved caspase-1, and GSDMD in cardiac tissues.However, JQ1 signi cantly inhibited LPS-induced up-regulation of NLRP3, Cleaved caspase-1, and GSDMD (Fig. 6b, c).In addition, we obtained the same results in H9C2 cells in vitro.In LPS-treated H9c2 cells, SIRT1 mRNA and protein levels were down-regulated, while JQ1 treatment upregulated SIRT1(Fig.6d, e).In LPS-treated H9C2 cells, protein expression levels of NLRP3, Cleaved caspase-1, and GSDMD were increased, while JQ1 treatment signi cantly reduced these changes (Fig. 6e,  f).These results suggest that JQ1 upregulates SIRT1 expression and inhibits NLRP3 in ammasome activation in vivo and in vitro.
3.6 SIRT1 antagonized the inhibitory effect of JQ1 on LPS-induced in ammation of H9C2 cells To further con rm whether JQ1 inhibits the release of in ammatory factors in LPS-treated mice and H9C2 cells through the SIRT1 pathway, SIRT1 inhibitor EX527 was added to the experiment.The results showed that, as described above, the release of IL-1β and IL-18 in LPS-treated H9C2 cells was signi cantly increased compared with the control group.JQ1 treatment inhibited the release of IL-1β and IL-18, whereas EX527 blocked the release of JQ1-mediated in ammatory cytokines (Fig. 7a, b).In addition, LPS-treated H9C2 cells were down-regulated in NLRP3, Cleaved caspase-1, and GSDMD protein expressions, while JQ1 treatment inhibited such changes.The addition of EX527 blocked the inhibitory effect of JQ1 on NLRP3 in ammasome activation (Fig. 7c-g).These results suggest that JQ1 may ameliorate LPS-induced cardiac in ammation through the SIRT1 pathway.

Discussion
In this study, we con rmed that NLRP3 in ammasome activation plays a vital role in the pathogenesis of LPS-induced cardiac dysfunction, which is consistent with previous studies [3,20,21,23].This study is the rst to demonstrate a protective effect of THE BRD4 inhibitor JQ1 on LPS-induced cardiac dysfunction.This study found that JQ1 signi cantly reduces cellular in ammation by inhibiting NLRP3 in ammasome activation in vivo and in vitro.In addition, we found that JQ1 increased SIRT1 expression in LPS-treated mice myocardium, and the inhibition of SIRT1 by EX527 partially eliminated the inhibition of JQ1 on NLRP3 in ammasome activation.This suggests that JQ1 inhibits the activation of NLRP3 in ammasome in mice by upregulating SIRT1, thereby protecting against LPS-induced cardiac dysfunction.
BRD4 inhibitor JQ1 was rst discovered because of its anti-tumor effect, and in recent years, its antiin ammatory effect has also been gradually discovered.In animal models, JQ1 treatment alleviates various in ammation, such as gastritis, osteoarthritis, pancreatitis, microglitis, etc. [4,5,10,26,35,38,42]In cardiovascular disease-related in ammation, JQ1 can alleviate myocardial infarction-induced in ammation by inhibiting the activation of TLR4 signal and the in ammatory response of vascular endothelial cells by inhibiting the MAPK/NF-κB signaling pathway [33,36].However, the role of JQ1 in LPS-induced cardiac dysfunction remains unclear.Therefore, we established a mice model of LPSinduced cardiac dysfunction and found that JQ1 could reverse cardiac dysfunction, restore the damage of cardiac tissue structure, reduce the in ammatory cell in ltration, reduce the release of serum myocardial enzymes (LDH, CK-MB) and in ammatory factors (IL-1β, IL-18), and reduce oxidative damage, and improve the survival rate.In addition, we obtained the same results in LPS-treated H9C2 cells.This suggests that JQ1 has a protective effect on LPS-induced cardiac dysfunction.
In the early stages of sepsis, a moderate in ammatory response can ward off foreign pathogens and prevent further tissue damage.However, excessive in ammation can lead to organ damage and dysfunction [9].NLRP3 in ammasome is thought to be involved in the in ammatory cascade ampli cation of sepsis and has a particular value in predicting the severity of sepsis patients [8,41].
Previous studies have suggested that NLRP3 in ammasome plays a vital role in maintaining normal cardiac function [43].When NLRP3 is activated, it is involved in the in ammatory response of the myocardium, secreting IL-1βand IL-18 and recruiting in ammatory cell in ltration, leading to myocardial injury [11].Our results showed that NLRP3, Cleaved caspase-1, and GSDMD protein levels were increased in vitro and in vivo models.However, JQ1 signi cantly reduced the levels of NLRP3, Cleaved caspase-1, and GSDMD, and inhibited the production of IL-1βand IL-18.Therefore, these data suggest that JQ1 protects against cardiac injury by inhibiting NLRP3 activation and in ammatory cytokine release.
SIRT1, a member of the sirtuin family, has histone deacetylase activity and can participate in many biological processes such as cell growth and apoptosis [2].In recent years, it has been found that the damage of the SIRT1 signaling pathway is one of the critical mechanisms mediating cardiac injury in sepsis, and activation of the SIRT1 signaling pathway is expected to become an essential strategy for the prevention and treatment of cardiac injury in sepsis [30].Han et al. found that compared with wild-type mice, the mortality and myocardial injury of SIRT1-/-mice were signi cantly increased after cecal ligation perforation [12].A recent study showed that the inhibition of BRD4 upregulates SIRT1 and restores impaired autophagy ow in an experimental model of acute pancreatitis[28].In the model of cardiac dysfunction induced by LPS in this experiment, we observed that JQ1 likewise increased SIRT1 levels in cardiac tissue and H9C2 cells.We further investigated whether SIRT1 is involved in the inhibitory effect of JQ1 on NLRP3 in ammasome activation in H9C2 cells.In the experiments, we found that JQ1 reversed the LPS-induced reduction in SIRT1 levels and inhibited the activation of the NLRP3 in ammasome.Inhibition of BET protein has not been reported to not only expose more acetylation sites for SIRT1 deacetylase activity but also upregulate SIRT1 levels, thereby enhancing SIRT1 deacetylation modi cation that in turn affect gene transcription induced during the in ammatory response.To further investigate the relationship between SIRT1 and NLRP3, we intervened using a speci c SIRT1 inhibitor of EX527 and showed that EX527 partially abolished the inhibitory effect of JQ1 on NLRP3 in ammasome activation.This suggests that SIRT1 may mediate the anti-in ammatory activity of JQ1 in LPS-induced cardiomyopathy.

Declarations AUTHOR CONTRIBUTION
All contributed to the study conception and design.Investigation, funding acquisition, data collection and analysis were performed by Fuyuan Liu and Rong Jiao.The rst draft of the manuscript was written by Wenjun Li and all authors commented on previous versions of the manuscript.All authors read and approved the nal manuscript.Figure 6

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