Kaixuan Qibi granules attenuate myocardial fibrosis through BRD4 blockade regulated by the NF-κB/NLRP3 signaling pathway

Abstract

Background and aim

Kaixuan Qibi granules (KXGs) have exhibited favorable therapeutic outcomes in addressing a wide range of ailments. They have established their efficacy as a traditional Chinese medicine formulation, particularly for cardiovascular and cerebrovascular diseases associated with myocardial fibrosis (MF). Targeting of bromodomain-containing protein 4 (BRD4) represents an efficacious strategy for addressing inflammation (a pivotal factor in MF progression). However, revealing the inhibitory impact of KXGs on BRD4 in MF development is elusive. We investigated the impact of KXGs on MF and elucidated the regulatory mechanism of BRD4 in MF by KXGs in vitro and in vivo.

Experimental procedure

Induction of an in vivo model of MF was achieved in C57BL/6 mice by intraperitoneal administration of isoproterenol (ISO) over 21 days. After administering different doses of KXGs and fosinopril (FOS) via oral gavage for 21 days, metabolite levels, myofibril accumulation, and collagen deposition were assessed using standardized assays. To simulate an inflammatory reaction in vitro, mouse monocyte–macrophage leukemia (RAW264.7) cells were stimulated with lipopolysaccharide (LPS). The intervention effect of KXGs on BRD4 and the nuclear factor-kappa B/NOD-like receptor protein 3 (NF-κB/NLRP3) pathway was evaluated using western blotting and immunofluorescence methods.

Results

In this study, we observed a notable reduction in the extent of fibrotic tissue accompanied by organized collagen following treatment with KXGs. KXGs exhibited a significant regulatory effect on metabolite levels while also inhibiting BRD4-related pathways and the inflammatory response. Later, KXGs exhibited downregulation the expression of BRD4, phosphorylated (p)-NF-κB p65 (phosphor/total), NLRP3, ASC, caspase-1, and interleukin-1β (IL-1β) in both in vitro and in vivo models of MF development.

Conclusion

KXGs attenuated MF progression by modulating the NF-κB/NLRP3 signaling pathway through inhibition of BRD4 activity.

Graphical abstract

1. Introduction

The prevalence of cardiovascular diseases (CVDs) makes them the leading cause of mortality globally, and a significant contributor to disability.¹ There has been a substantial increase in the global prevalence of CVDs, with the number of cases rising by 93 % from 271 million in 1990 to 523 million in 2019. In addition, mortality rates for CVD have witnessed an alarming surge of ∼54 %, escalating from 12.1 million deaths in 1990 to 18.6 million deaths in 2019, accounting for roughly one-third of annual global fatalities.²

The manifestation of myocardial fibrosis (MF) is observed commonly following the progression of various CVDs to an advanced stage.³ MF is characterized by a sudden onset, high mortality rates, and elusive underlying mechanisms. Thus, efficacious therapeutic interventions against MF are lacking.⁴ Relevant and effective therapeutic targets and treatment options are urgently warranted.

Bromodomain-containing protein 4 (BRD4) belongs to the bromodomain and extra-terminal (BET) protein family. BRD4 comprises two bromodomain domains and an interleukin (IL) domain that facilitate its functions as a genetic regulator and transcriptional cofactor. Investigations have substantiated the binding of BRD4 to the gene promoters of inflammatory cytokines in macrophages. This binding directly governs the modulation of inflammatory factors and corresponding immune reactions in these cells.⁵ Therefore, targeting BRD4 proteins represents a promising therapeutic strategy for numerous diseases. Recent studies have demonstrated the significant impact of the BET inhibitor JQ1 on cancer and inflammatory responses.^6,7 Another study demonstrated that inhibition of BRD4 expression mitigated the production of pro-inflammatory cytokines in microglia, thereby facilitating functional recovery following spinal-cord injury.⁸ In addition, inhibition of BRD4 expression has been shown to mitigate cardiomyocyte apoptosis through the nuclear factor-kappa B (NF-κB) pathway in a rat model of coronary occlusion.⁹

BRD4, by interacting with the NF-κB/NOD-like receptor protein 3 (NLRP3) signaling pathway, has a crucial role in regulating diverse biological processes, and contributes to the pathogenesis of inflammation and cancer. For example, inhibition of BRD4 expression has an antitumor effect in renal-cell carcinoma through activation of the NF-κB/NLRP3 pyroptosis signaling pathway.¹⁰ Studies conducted recently have revealed that the involvement of BRD4 on pyroptosis induced by crystals of monosodium uric acid is mediated through its regulation of the NF-κB/NLRP3/GSDMD signaling pathway, thereby presenting a promising target for the treatment of acute gouty arthritis.^11,12 A recent study indicated that inhibition of BRD4 expression exerted anti-inflammatory effects and analgesia against inflammatory pain by suppressing the activation of NF-κB and inflammasomes, thereby safeguarding neural cells from pyroptosis.¹³ Another study reported that inhibition of BRD4 expression mitigated matrix degradation in nucleus pulposus cells by augmenting autophagy and suppressing the activity of the NLRP3 inflammasome through the NF-κB signaling pathway.¹⁴ However, the precise relationship between BRD4 and inflammation associated with the NF-κB/NLRP3 signaling pathway in MF is unclear, which necessitates further investigation into the underlying molecular mechanisms.

Kaixuan Qibi granules (KXGs; license number: Z20240007000) is a pharmaceutical preparation approved by the Sichuan Provincial Drug Administration for use in medical institutions in China. KXGs are composed of angelica, rehmannia, peach kernel, safflower, aurantii, paeonia, bupleurum, glycyrrhiza, platycodon, rhizoma chuanxiong, achyranthes, stoloniferum, zedoary, allium, trichosanthes fructus bark, and cinnamon twig. Based on the Xuanfu theory,^{15, 16,15, 16} the original recipe has been utilized in clinical practice for nearly 30 years to address palpitation, dyspnea, chest tightness, and angina with remarkable therapeutic efficacy. The impact of fibrosis on cardiac function is widely acknowledged, yet there is a dearth of clinically approved targeted medications against MF.¹⁷ The key aspects of the definition of traditional Chinese medicine theory of MF include Yang deficiency and phlegm stasis, pervasive blood stasis, and Xuanfu occlusion.

Each KXG is formulated by combining Xuefu Zhuyu decoction and Gualou Xiebai Guizhi decoction, supplemented with zedoary and rhizoma sparganii while excluding magnolias. KXGs primarily target qi obstruction, phlegm turbidity, and blood stasis, with a focus on enhancing the blood circulation and resolving blood stasis. However, the regulatory impact of KXGs on BRD4 during MF has yet to be assessed. The underlying mechanism by which KXGs modulate BRD4 remains unexplored.

The objective of the present study was to investigate the inhibitory effect of KXGs on BRD4 expression in vitro and in vivo. Furthermore, we aimed to investigate the mechanistic basis through which KXGs interfere with BRD4 activity, thereby providing novel insights into their potential as therapeutic agents targeting cardiac fibrosis.

2. Materials and methods

2.1. Chemicals and reagents

KXGs (batch number: 20240115) were procured from the Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University (Luzhou, China). KXGs comprise a blend of 16 traditional Chinese herbs (Table 1). The standard compounds utilized for quality-control analysis (rehmanosine D, amygdalin, paeoniflorin, ferulic acid, licorice glycosides, verbascoside, naringin, neohesperidin, kaempferol) were obtained from the National Institutes for Food and Drug Control (Beijing, China). Fosinopril sodium (FOS; catalog number: HY-B0382) and isoproterenol hydrochloride (ISO; HY-B0468) were from MedChemExpress (Monmouth Junction, NJ, USA). Formic acid (high-performance liquid chromatography-grade), acetonitrile, and methanol were obtained from Thermo Fisher Scientific (Waltham, MA, USA). We acquired the following antibodies: BRD4 (28486-1-AP; Proteintech, Rosemont, IL, USA), phosphorylated (p)-NF-κB p65 (ab76302; Abcam, Cambridge, UK), NF-κB p65 (ab32536; Abcam), NLRP3 (ab263899; Abcam), ASC (12301-1-AP; Proteintech), caspase-1 (PA5-99390; Invitrogen, Carlsbad, CA, USA), IL-1β (16806-1-AP; Proteintech), fibronectin (15613-1-AP; Proteintech), collagen-І (AF7001; Affinity, Dallas, TX, USA), and collagen-ІІІ (22734-1-AP; Proteintech).

Table 1.

Prescription of Kaixuan Qibi granules.

English Name	Chinese Name	Scientific Name	Family	Part	Used Quantity (Dry Weight)
Peach kernel	Taoren	Prunus persica (L.) Batsch	Rosaceae	Seed	0.144 g
Safflower carthamus	Honghua	Carthamus tinctorius L.	Compositae	Dried flower	0.144 g
Peony	Chishao	Paeonia veitchii Lynch	Ranunculaceae	Roots	0.080 g
Rhizoma Chuanxiong	Chuan -xiong	Ligusticum chuanxiong Hort.	Apiaceae	Roots	0.144 g
rhizoma sparganii	Sanleng	Sparganium stoloniferum Buch. -Ham.	Sparganiaceae	Roots	0.144 g
Curcuma	Ezhu	Curcuma phaeocaulis Val.	Zingiberaceae	Roots	0.144 g
Achyranthes	Niuxi	Achyranthes bidentata Bl.	Amaranthaceae	Roots	0.144 g
Snakegourd	Gualou	Trichosanthes kirilowii Maxim.	Cucurbitaceae	Dried ripe fruit	0.080 g
Allium	Xiebai	Allium chinese G. Don	Liliaceae	Bulbus	0.080 g
Rehmannia	Dihuang	Rehmannia glutinosa Libosch	Scrophulariaceae	Roots	0.144 g
Angelica	Danggui	Angelica sinensis (Oliv.) Diels	Apiaceae	Roots	0.096 g
Platycodon	Jiegeng	Platycodon grandiflorum (Jacq.)A. DC.	Campanulaceae	Roots	0.048 g
Fructus aurantia	Zhiqiao	Citrus aurantium L.	Rutaceae	Dried ripe fruit	0.080 g
Bupleurum	Chaihu	Bupleurum chinense DC.	Apiaceae	Roots	0.048 g
Cassia	Gui Zhi	Cinnamomum cassia Presl	Lauraceae	Stem/Twig	0.032 g
Licorice	Gancao	Glycyrrhiza uralensis Fisch.	Fabaceae	Roots	0.048 g

2.2. Sample preparation for analyses by ultrahigh-performance liquid chromatography -high-resolution mass spectrometry (UPLC-HRMS)

The powdered form of KXGs was subjected to sonication with 50 × 75 % ethanol for 1 h to enable chemical profiling. The filtrate was subjected to centrifugation at an ultrahigh speed of 10,000×g for 15 min. The resulting supernatant was utilized for MS.¹⁸ Before the standard validation of the KXGs by UPLC-HRMS, nine standard compounds (rehmanosine D, amygdalin, paeoniflorin, ferulic acid, licorice glycosides, verbascoside, naringin, neohesperidin, kaempferol) were dissolved in methanol.

2.3. UPLC-HR-MS conditions for comprehensive chemical characterization

KXGs were chemically characterized using an ultrahigh performance liquid chromatography system (UltiMate™ 3000), which was coupled with a high-resolution mass spectrometer (Q-Exactive) through an electrospray ionization interface, all provided by Thermo Fisher Scientific. The chromatography system comprised an autosampler, column compartment, dual pumps, and diode array detector. The KXGs extract was separated using an Acquity® UPLC BEH C₁₈ column (1.7 μm, 2.1 × 100 mm; Waters, Waltham, MA, USA) under a column temperature of 40 °C. The chromatographic parameters were optimized. The elution gradient was established using a mixture of water containing 0.1 % formic acid (A) and acetonitrile (B), with a flow rate set at 0.2 mL/min. The gradient was programmed as follows: 98 % A for 0–5 min; 88 % A for 5–8 min; 75 % A for 8–35 min; 20 % A for 35–50 min; 0 % A for 50–58 min; and 95 % A for 58–60 min. The autosampler temperature was maintained at 4 °C throughout analyses, while the injection volume remained fixed at 5.0 μL.^19,20

Negative full-scan mode was employed to precisely capture molecular ions within the range of m/z 70–1050 Da at a resolution of 70,000, thereby facilitating the identification of components in the KXGs extract. Additional settings comprised a spray voltage of −3.0 kV, auxiliary gas flow rate of 10 arb, sheath gas flow rate of 35 arb, capillary temperature set at 320 °C, vaporizer temperature maintained at 250 °C, and an RF lens adjusted to 50 %. Standards were employed for assistance of component identification. The control and data handling for UPLC-HRMS were managed using software (Xcalibur 2.2 SP1) from Thermo Fisher Scientific.²¹

2.4. Animals and drug administration

Male C57BL/6J mice (22 ± 2 g; 8–10 weeks) were procured from GemPharmatech (Chengdu, China) and subjected to a 1-week acclimatization period. The experimental procedures and animal welfare were in accordance with the Guide for the care and use of laboratory animals (US National Institutes of Health, Bethesda, MD, USA). The study protocol was approved (20231218-018) by the Animal Care and Use Committee of Southwest Medical University. Mice were allowed to acclimatize in a room at 22 ± 0.5 °C and relative humidity of 60 ± 5 %, and exposed to a 12-h light/dark cycle. They were provided ad libitum access to a standard diet and water for drinking while receiving humane treatment.

Fifty mice were allocated randomly into two groups: normal control (NC), who received a standard diet, and four other groups (each consisting of 10 mice) who underwent intraperitoneal injection of ISO (10 mg/kg) once-daily for 21 days to induce MF. Furthermore, ISO-injected mice were allocated randomly to four groups: ISO, low-dose KXGs (KX-L; 34 mg/kg), high-dose KXGs (KX-H; 68 mg/kg), and positive control (treated with FOS at 10 mg/kg). The prepared KXGs extract was administered twice-daily via the oral route to mice suffering from MF at 0.15 mL/100 g bodyweight for an additional 21 days. Parameters of cardiac function were assessed on day 21 using an ultra-high-resolution small-animal ultrasound imaging system (Vevo 3100; Fujifilm VisualSonics, Toronto, Canada). The left ventricular end-systolic diameter (LVESD), fractional shortening (FS%), and ejection fraction (EF%) were computed.

2.5. Untargeted metabolomics analysis

On day 21, mice were anesthetized with 3 % isoflurane and blood samples were collected from the orbital vein. Samples were collected and stored at room temperature for 1–2 h. Subsequently, the serum was isolated by centrifugation (6000×g, 10 min, 4 °C), followed by storage at −80 °C. Finally, employing the Orbitrap principle, the metabolites in the samples were detected using high-resolution liquid chromatography-tandem mass spectrometry. Then, these metabolites were matched qualitatively and quantitatively with information from a local database (www.hmdb.ca), including retention time, molecular mass, secondary spectrum, and other relevant characteristics.²² The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation information for these metabolites was obtained from the Lipid Metabolites and Pathways Strategy (LIPID MAPS, www.lipidmaps.org/) and KEGG databases (www.genome.jp/kegg/pathway.html/). Enrichment analysis using the hypergeometric distribution test was conducted to compare the distribution of KEGG pathways between differential metabolites and total metabolites.

2.6. Staining

Sections of thickness 4 μm were cut from paraffin-embedded tissues and underwent routine staining (hematoxylin and eosin (H&E), Masson's trichrome, Sirius red) according to published methods.¹⁹ Stained sections were examined under a digital pathology scanner (Konfoong Bioinformation Tech, Nibo, China).

2.7. Immunofluorescent staining of myocardial-tissue specimens

For immunofluorescence staining, cryosections of myocardial-tissue specimens of thickness 4 μm were prepared meticulously and immersed subsequently in a solution containing 4 % paraformaldehyde. Sections were initially fixed with 5 % goat serum, followed by treatment with primary antibodies and incubation overnight at 4 °C. Subsequently, slides were washed thrice with phosphate-buffered saline for 10-min each before being exposed to secondary antibodies for 50 min. Sections were counterstained with 4′,6-Diamidino-2-phenylindole dihydrochloride (C0065; Solarbio, Beijing, China) for 10 min. Subsequently, they were rinsed with phosphate-buffered saline, preserved in 20 % glycerol solution, and captured using a fluorescence inverted microscope (EVOS; Thermo Fisher Scientific).

2.8. Western blotting

Protein extraction was carried out using ice-cold RIPA lysis buffer (BL504A; Biosharp, Hefei, China). Protein quantification was done using the bicinchoninic acid assay. Protein samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride (PVDF) membranes (0.25 μm; IPVH00010; Merck Millipore, Burlington, MA, USA). Following 1-h incubation at room temperature with 5 % blotting grade solution, primary antibodies (ASC, 1:1000 dilution; BRD4, 1:1000; caspase-1, 1:1000; IL-1β, 1:1000, NLRP3, 1:1500, NF-κB p65, 1:1000, p-NF-κB p65, 1:1000; glyceraldehyde 3-phosphate dehydrogenase, 1:3000) were added and left overnight at 4 °C. After washing, secondary antibodies labeled with horseradish peroxidase were incubated at 37 °C for 1 h. Protein bands were visualized utilizing the Super ECL blotting method (BG0001; BB Biotech, Schaffhausen, Switzerland).

2.9. Cytotoxicity assay and cell experiments

Mouse monocyte–macrophage leukemia (RAW264.7) cells were obtained from Cellverse Bioscience Technology (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) supplemented with 1 % penicillin-streptomycin (Gibco) and 10 % fetal bovine serum (Gibco), and incubated at 37 °C in an atmosphere of 5 % CO₂. RAW264.7 cells were cultured in a 96-well plate at a density of 5 × 10³ cells per well. Subsequently, the cells were incubated for 48 h overnight. Subsequently, 100 μL of CellTiter-Lumi™ Plus (Beyotime, Shanghai, China) detection reagent was added to each well and agitated for 5 min to ensure complete cell lysis. The luminescence (RLU) of each well was measured using a multipurpose microplate reader equipped with a luminometer. The absorbance values of the test wells were recorded as RLU treatment, those of the negative controls RLU controls, and the blank values as RLU blank for each assay. The inhibition rate (%) was calculated using the following formula: (RLU_control − RLU_blank − RLU_treated)/(RLU_control − RLU_blank) × 100. Half maximal inhibitory concentration values were determined using Paradigm 9 (GraphPad, La Jolla, CA, USA).

In addition, RAW264.7 cells were cultured to a density of 50 % and subsequently starved in a medium containing 3 % fetal bovine serum for 12 h prior to stimulation with lipopolysaccharide (LPS) for 48 h. Protein expression of ASC, BRD4, caspase-1, NLRP3, NF-κB p65, p-NF-κB p65, and IL-1β in RAW264.7 cells treated with varying concentrations of KXGs for 24 h was measured using western blotting.

2.10. Statistical analyses

Statistical analysis was conducted using GraphPad Prism 9 software. Statistical significances were determined through Two-way ANOVA tests. Experimental values are presented as the mean ± standard deviation, derived from a minimum of three independent experiments.

3. Results

3.1. Qualitative identification of compounds in KXGs and prediction of their target activity

After optimization of chromatographic conditions, UPLC-HRMS was employed for compound identification in KXGs (Fig. 1A). Nine compounds (rehmanosine D, amygdalin, paeoniflorin, ferulic acid, liquiritin, verbascoside, naringin, neohesperidin, and kaempferol) were confirmed to be in KXGs according to comparison with standards (Fig. 1B and C). Further target analysis of the active constituents present in KXGs revealed 168 targets that exhibited significant associations with inflammation (Fig. 1D), suggesting that KXGs could ameliorate the inflammation observed in MF. The underlying mechanism of KXGs in the management of MF remains unclear.

Fig. 1.

Qualitative analysis of KXGs using UPLC-HRMS. (A) UPLC-HRMS chromatogram of KXGs in negative-ion mode. (B–C) UPLC-HRMS chromatogram of nine reference standards, namely rehmanosine D (1), amygdalin (2), paeoniflorin (3), ferulic acid (4), liquiritin (5), verbascoside (6), naringin (7), neohesperidin (8), and kaempferol (9). (D) Identification of 168 KXGs targets closely associated with myocardial fibrosis-related inflammation.

3.2. KXGs mitigated ISO-induced MF

MF is commonly observed in various CVDs, and often occurs concurrently with impaired cardiac physiological function.²³ To investigate the therapeutic efficacy of KXGs on MF, we used an ISO model that mimics myocardial infarction in mice. Mouse heart samples were harvested and analyzed (Fig. 2A). In comparison with the NC group, accumulation of myocardial myofibrils was observed in the heart tissues of ISO-group mice, which were stained with H&E; however, significant pathologic alterations were not detected in the liver, spleen, lungs, or kidneys (Fig. 2B). Furthermore, a significant reduction in fibrotic area with well-organized myofibrils was observed following treatment with KXGs compared with that in the ISO group (Fig. 2B). These findings suggested that KXGs could alleviate ISO-induced MF, while the ISO model did not exhibit evident pathological changes in other major organs.

Fig. 2.

KXGs exhibited remarkable efficacy in inhibiting ISO-induced MF while demonstrating no pathologic alterations in other organs. (A) Portrayal of the procedural framework involved in establishing the model of MF in C57BL/6 mice and evaluating the therapeutic efficacy of KXGs. (B) Sections of liver, heart, spleen, lung, and kidney tissue underwent histology with H&E staining (n = 4). The black arrow in the ISO group indicates the region of myocardial disarray with inflammatory infiltration and fibrotic changes. Scale bars = 100 μm.

3.3. KXGs suppressed the progression of collagen deposition in vivo during MF

The therapeutic efficacy of KXGs was evaluated by establishing and applying ISO-induced MF. Echocardiography was utilized to assess cardiac function (Fig. 3A), which confirmed establishment of the MF model. Mice with ISO-induced MF demonstrated a significant deterioration in cardiac function, as characterized by a notable decrease in EF% and FS%, along with elevation of LVESD (Fig. 3B−D). Furthermore, to assess the potential impact of KXGs on collagen deposition, we conducted staining (Masson's trichrome, Sirius red, immunofluorescence) in myocardial tissue samples from mice. We revealed a notable reduction in the extent of fibrotic tissue accompanied by organized collagen following treatment with KXGs as compared with that in the ISO group, suggesting that KXGs could alleviate the MF induced by ISO (Fig. 3E and F). Taken together, the administration of KXGs suppressed the progression of collagen deposition in vivo during MF, though its targets require further elucidation.

Fig. 3.

KXGs suppressed the progression of collagen deposition in vivo during MF. (A) Representative echocardiography graphs of NC, ISO, KX-L, KX-H, and FOS groups. Echocardiography was utilized for quantitative assessment of impaired cardiac function through (B) EF%, (C) FS%, and (D) LVESD (mm). (E) Myocardial tissue sections were subjected to staining (Masson's trichrome, Sirius red). Black arrows indicate collagen fibers (blue in Masson staining; red in Sirius Red staining). Scale bar: 100 μm. (F) The MF induced by ISO was assessed through immunofluorescent staining against collagen-І (green), collagen-ІІІ (green), and fibronectin (green), with counterstaining using DAPI (blue: nuclei) in the experimental groups indicated. Scale bar: 100 μm. Data are the mean ± SD (n = 4). Statistical significance was observed at p < 0.05∗ and p < 0.01∗∗. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. Untargeted metabolomics analysis

The application of untargeted metabolomics analysis is a crucial approach for investigating the impact of diverse treatments on metabolites and elucidating inflammation-related mechanisms and targets. We conducted a comparative analysis of the metabolites in serum treated with a standard diet (NC), ISO, and KXGs (68 mg/kg). The Venn diagram depicted in Fig. 4A showcases remarkable disparities in the metabolites among the three treatment cohorts. The scatter colors represent the distinct experimental groupings. The scores for orthogonal projections to latent structures-discriminant analysis (OPLS-DA) clearly demonstrated an important distinction between the ISO group and the KXGs group (Fig. 4B). Volcano plots depicted a comprehensive distribution of different metabolites (Fig. 4C). Most analytes were portrayed as gray, signifying their lack of statistical significance. Among the 406 metabolites, 170 had upregulated expression and 236 had downregulated expression, and exhibited significant differences. Subsequently, employing heatmaps based on the corresponding peak area, we conducted a comparative assessment of metabolite levels between the ISO group and KXGs to ascertain their relative abundances (Fig. 4D). Analyses of signaling-pathway enrichment using the KEGG database provided additional evidence for the significant participation of these metabolites in “Kaposi sarcoma-associated herpesvirus infection” and “Sulfur relay system” (Fig. 4E). These findings suggested that KXGs therapy exerted a significant regulatory effect on metabolite levels, inhibited BRD4-related pathways and, ultimately, suppressed the inflammatory response.

Fig. 4.

Untargeted metabolomics analysis of serum following various treatments. (A) Venn diagrams depicting the metabolites of distinct treatment cohorts. (B) OPLS-DA of serum metabolites under different cohorts. (C) Volcano plots illustrating the differential abundance of metabolites in serum subjected to various treatments. (D) Heatmaps displaying significant variations in serum metabolite profiles among different treatment cohorts. (E) Bubble diagram depicting the enrichment of differential metabolites in cell metabolism-related pathways based on the KEGG database.

3.5. KXGs inhibited the development of LPS-induced activation of inflammatory cytokines in vitro

The Cell Counting Kit-8 assay was used to evaluate the effect of KXGs on the viability of RAW264.7 cells. In the range of 0.125–2.0 mg/mL KXGs concentration, the survival rate of RAW264.7 cells was about 100 %, demonstrating minimal cytotoxicity (Fig. 5A). The inhibitory effect of KXG particles on the activation of inflammatory factors was validated in a macrophage cell line. Due to its ability to enhance inflammation and fibrosis, LPS is employed frequently in vitro for simulating the inflammatory process induced by MF. We consistently detected changes in BRD4 expression within RAW264.7 cells following 48-h exposure to LPS. Moreover, in comparison with the NC group, LPS upregulated the expression of ASC, caspase-1, NLRP3, p-NF-κB p65, and IL-1β, whereas KXGs could downregulate the expression of BRD4, ASC, caspase-1, NLRP3, p-NF-κB p65 (phosphor/total), and IL-1β. The curative effect of the particle concentration was simultaneously presented in a dose-dependent relationship by KXGs, exhibiting an optimal concentration of 1 mg/mL (Fig. 5B–H). These results indicated that, in vitro, KXGs could impede the inflammatory process.

Fig. 5.

KXGs inhibited the development of LPS-induced activation of inflammatory cytokines in vitro. (A) Cell viability was evaluated following a 48-h incubation of RAW264.7 cells with varying concentrations of KXGs. (B–H) Expression of proteins related to inflammatory cytokines, such as BRD4, ASC, caspase-1, NLRP3, NF-κB p65 activation index (p-NF-κB p65/NF-κB p65 ratio), and IL-1β, after KXGs treatment, were compared among the LPS group and KXGs group in RAW264.7 cells.

3.6. Impact of KXGs on MF in vivo

The aforementioned findings showed that stimulation with LPS led to marked upregulation of expression of BRD4 and its downstream effectors NF-κB and NLRP3 in RAW264.7 cells, but treatment with KXGs counteracted the LPS-induced upregulation of these factors. However, the underlying mechanisms were not known. To further validate the efficacy of KXGs in regulating the BRD4/NF-κB/NLRP3 signaling pathway, we conducted an in vivo experiment to evaluate the intervention effect exerted by KXGs on this signaling cascade. The ISO group exhibited upregulation of expression of ASC, BRD4, NLRP3, and p-NF-κB p65 (Fig. 6A), indicating the presence of an inflammatory response. In comparison with the ISO group, the KX-L group and KX-H group had reduced expression of BRD4, ASC, NLRP3, and p-NF-κB p65. These data further demonstrated that KXGs could elicit inhibitory effects on signal transduction associated with inflammation and pyroptosis during fibrosis progression. The NLRP3 inflammasome comprises proinflammatory factors (NLRP3, ASC, pro-caspase-1), which undergo assembly and activation upon signal stimulation.²⁴ Then, the induction of IL-1β and caspase-1 disrupts the integrity of the cellular membrane, ultimately leading to pyroptosis.²⁵ In the present study, immunofluorescence analysis of IL-1β and caspase-1 revealed significant upregulation in the activation of pyroptosis pathways in ISO-induced MF. Treatment with KXGs led to remarkable suppression of expression of IL-1β and caspase-1, surpassing that of the ISO group in comparison (Fig. 6B). Subsequently, protein expression of ASC, BRD4, NLRP3, NF-κB p65, p-NF-κB p65, caspase-1, and IL-1β in myocardial tissues was quantified by western blotting. The expression of proteins related to inflammatory factors was decreased significantly following administration of KXGs (Fig. 6C–I), and was consistent with the results obtained from immunofluorescence staining. The aforementioned results suggested that KXGs exerted a robust intervention effect on the BRD4/NF-κB/NLRP3 signaling pathway during MF progression (see Fig. 7).

Fig. 6.

KXGs inhibit the BRD4/NF-κB/NLRP3 signaling pathway during MF in vivo. (A) Immunofluorescence staining of ASC (green), BRD4 (green), NLRP3 (green), and p-NF-κB p65 (green) in myocardial tissue sections (n = 4). Scale bar: 100 μm. (B) Immunofluorescence staining of the proteinase caspase-1 (green) and inflammatory factor IL-1β (green) in myocardial tissue sections (n = 4). Scale bar: 100 μm. (C–I) Protein expression of ASC, BRD4, NLRP3, NF-κB p65 activation index (p-NF-κB p65/NF-κB p65 ratio), caspase-1, and IL-1β in myocardial tissue was assessed using western blotting, Data are the mean ± SD (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7.

Schematic of the potential mechanism underlying the impact of KXGs on attenuating MF. KXGs, comprising 16 traditional Chinese herbs, were found to consist of nine components by UPLC-HRMS, and these components have anti-inflammatory potential. Intraperitoneal injection of ISO in C57BL/6J mice elicited macrophage activation, which involved the BRD4/NF-κB/NLRP3 signaling pathway and triggered the secretion of a multitude of inflammatory mediators. KXGs inhibited the expression of BRD4 protein and subsequently modulated the NF-κB/NLRP3 signaling pathway, thereby attenuating inflammation and MF.

4. Discussion

As a member of the BET family of epigenetic regulatory proteins, BRD4 engages in interactions with diverse protein partners. BRD4 has pivotal roles in the inflammatory response, apoptosis, and cell proliferation.²⁶ However, despite being an intriguing target for inflammation-related conditions,^{27, 28, 29} the precise molecular mechanisms underlying the potential clinical benefits of targeting BRD4 for MF treatment remain elusive and warrant further investigation.

We disclosed the crucial signaling pathway that unveils the BRD4-mediated modulation of MF. We revealed significant upregulation of BRD4 expression in macrophages stimulated by LPS and cells with MF induced by ISO. The findings presented here are in accordance with data from previous studies,^{30, 31, 32} and provide further evidence for the crucial role of BRD4 in regulating the development of MF across various conditions. Importantly, we revealed significant upregulation of BRD4 expression in MF progression upon treatment with KXGs.

CVDs encompass a spectrum of complex clinical manifestations, including myocardial fibrosis, myocardial hypertrophy, and heart failure. The underlying pathological mechanisms involve a diverse array of molecular and cellular processes, with the interplay between autophagy and inflammasome activation playing a pivotal role in cardiovascular pathophysiology.^33,34 For instance, in heart failure, alterations in autophagy levels are intricately linked to cardiomyocyte injury and apoptosis.³⁵ Research has demonstrated that autophagy exhibits distinct patterns across different stages of heart failure, characterized by reduced autophagy during the initial phase of cardiac hypertrophy and a pronounced increase in cardiac tissue during the advanced stages of heart failure.³⁶ Mitophagy, a specialized subtype of autophagy, is instrumental in preserving mitochondrial and cardiac functionality within cardiomyocytes by selectively degrading dysfunctional mitochondria, thereby protecting these cells from cellular damage. In the realm of neuroinflammation, autophagy exerts a substantial impact, alleviating neuroinflammation and neural injury through a variety of proteins associated with autophagy. Notably, autophagy functions as an inhibitory modulator of NLRP3-mediated inflammation in acute ischemic stroke, dampening inflammation by sequestering mitochondrial-derived reactive oxygen species that trigger the NLRP3 inflammasome. For instance, autophagy-related proteins such as Beclin-1, LC3, and p62 have been extensively researched for their inhibitory effects on NLRP3 inflammation by facilitating alkaline mitophagy.³⁷ Furthermore, a multitude of Traditional Chinese Medicine compounds may modulate the NLRP3 inflammasome by inducing the activation of autophagy.^38,39 Based on untargeted metabolomics analysis, we observed that KXGs exerted significant regulatory effects on metabolic profiles. In addition, enrichment analyses of these metabolites revealed their distinct involvement in the “Sulfur relay system” and “Kaposi sarcoma-associated herpesvirus infection pathways”. Notably, these signaling pathways are closely associated with inflammation⁴⁰ and the BRD4 protein,^{41, 42,41, 42} respectively.

The BRD4 protein has a crucial role in regulating the immune response and inflammatory responses by modulating the NF-κB/NLRP3 pathway. The latter encompasses the activation of inflammasomes as well as modulation of gene expression that impacts various biological processes. Studies have shown BRD4 to be implicated in a noncanonical epigenetic paradigm in atherosclerosis, which is activated via the STING-PERK pathway, resulting in aberrant activation of NF-κB and IRF3.⁴³ The transcription factor NF-κB plays a pivotal part in the modulation of genes associated with the immune response and inflammation.⁴⁴ NF-κB binds specifically to the promoter region of NLRP3, and exerts precise control over its transcriptional activity.⁴⁵ BRD4 is a pivotal mediator in the NF-κB/NLRP3 signaling pathway and a vital modulator of inflammation. It has been demonstrated to modulate the NF-κB/NLRP3 pathway induced by monosodium urate crystals in gouty arthritis and mitigate the inflammatory response associated with this condition.⁴⁶ Hence, regulating BRD4-mediated signaling pathway could be a promising therapeutic approach for inhibiting inflammation in MF using KXGs. This finding was validated through in vitro and in vivo experiments, where KXGs suppressed activation of NF-κB/NLRP3 signaling significantly during MF.

KXGs are employed to disperse excessive Yang, resolve phlegm, eliminate blood stasis, and facilitate the circulation of Qi. They are indicated for the treatment of symptoms including palpitations, dyspnea, chest constriction, and thoracic pain. The collective findings presented here offer compelling evidence to support the inhibitory effect of KXGs on BRD4 by modulating the NF-κB/NLRP3 signaling pathway, thereby preventing MF development. In conclusion, KXGs are expected to serve as a foundation for further research, potentially leading to the discovery of novel Chinese medicines aimed at treating MF.

Data availability

The data supporting the findings of this study are provided in the article. The supplementary data can be obtained upon reasonable request from the corresponding author.

Ethics statement

This study was carried out in accordance with the recommendations of regulations of the experimental animal ethics committee of Southwest Medical University. The protocol was approved by the experimental animal ethics committee of Southwest Medical University (No.20231218−018).

Funding acknowledgments

This is an open project of State Key Laboratory of Quality Research in Chinese Medicine funded by Macau Science and Technology Development Fund (Macau University of Science and Technology, 006/2023/SKL), Macau Special Administrative Region. This study was funded by the Science and Technology Development Fund, Macau SAR (File no. 0105/2022/A2), Luzhou-Southwest Medical University cooperation project (Grant Number: 2021LZXNYD-P02 and 2024LZXNYDJ060), the collaborative innovation task of Sichuan Science and Technology Program (Grant Number: 2022YFS0618-B2), Sichuan Administration of Traditional Chinese Medicine (Grant Number: 2021DZ05), Luzhou Science and Technology Program (Grant Number: 2022-JYJ-104), Southwest Medical University Technology Program (Grant Number: 2024ZXYZX04).

Declaration of competing interest

The authors declare that there are no conflicts of interest associated with the manuscript.

Abbreviations:

KX granules

Kaixuan Qibi granules

MF

myocardial fibrosis

BRD4

bromodomain-containing protein 4

ISO

isoproterenol

KX-L

Low-dose KX

KX-H

high-dose KX

FOS

Fosinopril

RAW264.7

Mouse Monocyte-macrophage Leukemia Cells

LPS

lipopolysaccharide

CVDs

Cardiovascular diseases

BET

Bromodomain and Extra-Terminal

UPLC-HR-MS

ultra-high performance liquid chromatography-high-resolution mass spectrometer

NC

regular diet

EF%

ejection fraction

FS%

fractional shortening

LVESD

left ventricular end-systolic diameter

H&E

hematoxylin and eosin

OPLS-DA

orthogonal projections to latent structures-discriminant analysis

KEGG

Kyoto Encyclopedia of Genes and Genomes