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Parthenolide improves sepsis-induced coagulopathy by inhibiting mitochondrial-mediated apoptosis in vascular endothelial cells through BRD4/BCL-xL pathway

Journal of Translational Medicine volume 23, Article number: 80 (2025) Cite this article

Abstract

Background

Sepsis is a systemic inflammatory syndrome that can cause coagulation abnormalities, leading to damage in multiple organs. Vascular endothelial cells (VECs) are crucial in the development of sepsis-induced coagulopathy (SIC). The role of Parthenolide (PTL) in regulating SIC by protecting VECs remains unclear.

Methods

The study utilized septic rats and lipopolysaccharide (LPS)-stimulated VECs to simulate a SIC model and observe the therapeutic effects of PTL. Additionally, nanotechnology was employed to produce Nano-PTL (N-PTL), to observe whether it has advantages over PTL in treating SIC.

Results

PTL has been shown to mitigate lung injury in septic rats, significantly reduce tumor necrosis factor-α (TNF-α) levels, and increase survival rates. PTL treatment also enhances coagulation function, augments vascular endothelial cell (VEC) function, reduces mitochondrial fragmentation, and increases both mitochondrial oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP), while inhibiting reactive oxygen species (ROS) production. By increasing BRD4/BCL-xL levels, PTL can prevent mitochondrial-mediated apoptosis in VECs, improve VEC function, and consequently ameliorate SIC. Additionally, nanotechnology-synthesized N-PTL further enhances the protective effects on VECs and coagulation function.

Conclusions

This study clarifies the therapeutic effects and mechanisms of PTL on SIC, offering new strategies and directions for the treatment of sepsis.

Introduction

Sepsis represents a systemic inflammatory response syndrome, characterized by the induction of inflammatory reactions, aberrant immune responses, and coagulation abnormalities, leading to multi-organ damage [1, 2]. Sepsis-induced coagulopathy (SIC) represents a severe and complex pathological condition, characterized by excessive activation and dysregulation of the coagulation system, which can lead to disseminated intravascular coagulation. Unfortunately, nearly all clinical studies focused on the prevention and treatment of SIC have been unsuccessful [3,4,5,6]. Therefore, it is essential to conduct in-depth research on the pathogenic mechanisms of SIC and to develop novel therapeutic strategies.

The vascular endothelium acts as a critical barrier between the blood and tissues, serving as a primary pathway for the dissemination of bacteria and endotoxins. It is also a vital organ involved in regulating blood flow, inflammation, and coagulation [7]. Vascular endothelial cells (VECs) are crucial in the progression of SIC. Infection and the inflammatory response can impair endothelial barrier function, facilitating the entry of bacteria and toxins into the bloodstream. Consequently, the damaged VECs activate the coagulation system, increasing the production of coagulation factors and thrombin activity, which leads to thrombosis. Simultaneously, they can promote leukocyte and platelet activation and aggregation, exacerbating inflammation and causing microvascular obstruction [8]. Additionally, damage to VECs may trigger the release of a cascade of cytokines and inflammatory mediators, further activating the coagulation system [9]. Therefore, the protection and repair of VECs may serve as a significant therapeutic strategy for SIC.

Mitochondria function as the central hub for cellular energy metabolism and the regulation of cell death [10]. Mitochondrial dysfunction is closely linked with endothelial cell damage. It can lead to an insufficient energy supply, resulting in endothelial cells being unable to perform essential functions, such as vascular relaxation, thrombosis inhibition, blood flow regulation, and the prevention of inflammatory responses [11, 12]. Moreover, mitochondrial dysfunction can exacerbate the endothelial cells’ response to oxidative stress, a critical factor in endothelial cell damage and vascular inflammation [13, 14]. Additionally, mitochondria release components such as cytochrome C, which can activate apoptotic signals, promote cell apoptosis, increase tissue factor expression, and enhance the activation of the exogenous coagulation system [15].

Parthenolide (PTL), a sesquiterpene lactone derived from plants in the asteraceae family, has been shown to possess anti-inflammatory and antitumor properties. Notably, it exhibits a significant inhibitory effect on nuclear factor-kappa B (NF-κB), as demonstrated by previous studies [16, 17]. PTL is considered to have therapeutic potential for sepsis by inhibiting NF-κB activity, thereby reducing the production and release of inflammatory mediators and providing protection to organs affected by sepsis [18]. Previous findings suggested that PTL can suppress the damage to the intestinal barrier in sepsis by inhibiting 5-hydroxytryptamine receptor 2A (5-HTR2A) [19]. In addition, PTL can also regulate the immune microenvironment in sepsis [20]. Furthermore, PTL has been found to inhibit the activation and aggregation of platelets [21]. However, it remains unclear whether PTL can ameliorate SIC through the protection of VECs.

Monomeric traditional Chinese medicines have shown potential in treating various diseases; however, their clinical application is limited by pharmacokinetic properties such as low bioavailability and poor stability. Nanotechnology, which transforms drugs into nanoparticles, has been demonstrated to improve pharmacological properties such as solubility, stability, and bioavailability [22]. Moreover, nanomedicines can increase stability and extend the half-life within the body, thereby enhancing therapeutic efficacy [23]. Current research has confirmed that nano-PTL (N-PTL) effectively protects intestinal barrier function in sepsis [19]. Nonetheless, additional studies are required to ascertain whether N-PTL can effectively treat SIC.

This study employed septic rats and lipopolysaccharide (LPS)-stimulated VECs to model sepsis and investigate the therapeutic effects of PTL on VECs and SIC. Furthermore, nanotechnology was used to develop N-PTL to evaluate whether N-PTL offers advantages over PTL in treating SIC. The objective is to identify new targets and strategies for the treatment of sepsis patients.

Materials and methods

Ethics statement

Experiments involving animals were conducted in strict adherence to the guidelines provided by the Animal Research: Reporting of In Vivo Experiments (ARRIVE). The procedures were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (Approval No. AMUWEC20237118).

Reagents

PTL (HY-N0141) and B-cell lymphoma-extra large (BCL-xL) inhibitor A-1331852 (HY-19741) were purchased from MedChemExpress (USA). ELISA kit of TNF-α (E-EL-R2856c) was purchased from Elabscience company (China). Antibodies for bromodomain-containing protein 4 (BRD4) (A18840) and β-actin (AC004) were purchased from Abclonal (China). Antibodies for BCL-xL (ab32370) was purchased from Abcam (USA). Antibodies for E-selectin (bs-1273R), Thrombomodulin (bs-0525R) and VE-Cadherin (bs-0878R) were purchased from Bioss (China). VE-Cadherin (36–1900) was purchased from Invitrogen (USA). LPS (L4130) was purchased from Sigma (USA). JC-1 (C2003S) and DCFH-DA detection kits (S0033) were purchased from Beyotime Biotechnology (China). MitoBright-Deep Red (MT12) was purchased from Dojindo (Japan). In situ cell death detection kit (11684795910) were purchased from Roche (Switzerland). Adenoviral vectors for BRD4 and BCL-xL deletion (Ad-shBRD4 and Ad-shBCL-xL) were generated by Obio life Technology (China). The target sequences were as follows: BRD4 shRNA, 5′-CCAAGAGGCAGACCAACCA-3′, Bcl-xL shRNA, 5′-GGGAGCTGGTGGTTGACTTTC-3′, All other chemicals were purchased from Sigma unless specifically mentioned.

Animal management and sepsis model establishment

A total of 90 adult Sprague–Dawley rats (200–220 g) from Army Medical University were kept in a facility with positive-pressure ventilation, free access to food and water, and conditions of 23–25 °C temperature and 40–70% humidity. The Sham group only underwent an abdominal incision and closure. Previous research protocols were referenced to establish a sepsis model of rats, using the method of cecal ligation and puncture (CLP) [24]. 30 min before CLP establishment, PTL (5 mg/kg) was given via tail vein. 24 h after the CLP model was established, blood and lung tissues were extracted from the rats. 18 (20%) rats were randomly divided into 3 groups: Sham, Sepsis, and Sepsis + PTL, with 6 rats in each group, for coagulation function, inflammatory factors, HE staining, and immunohistochemistry. 48 (53.3%) rats were randomly divided into three groups: Sham, Sepsis, and Sepsis + PTL, with 16 rats in each group, for survival analysis. 12 (13.3%) rats were randomly allocated into 2 groups: Sepsis + PTL and Sepsis + PTL + A-133, with 6 rats per group, to assess coagulation function and immunohistochemistry. 12 (13.3%) rats were randomly divided into 2 groups: LPS + PTL and LPS + N-PTL, with 6 rats in each group, for coagulation function and immunohistochemistry.

Tail bleeding time test

As described in our previous study [25], a rat tail marked with a 3 mm radius was soaked in 37 °C saline in a 15 mL tube for 2 min. After removal, a vertical incision was made at the mark, severing the tail, which was then placed back in the saline. Bleeding time was recorded, and the experiment ended after 20 min regardless of bleeding status. The wound was sutured with 4-0 Vicryl and cauterized with a silver nitrate stick to prevent further bleeding.

Multiplex immunohistochemistry and multichannel imaging

Tissue sections were deparaffinized using xylene and rehydrated through a graded series of alcohol solutions. Antigen retrieval was carried out with AR6 buffer (Akoya Biosciences) in a microwave oven. To inactivate endogenous peroxidase, slides were incubated with 3% H2O2 for 10 min. Multiplex immunohistochemistry was conducted over several staining rounds, each involving a protein block with 1% BSA, followed by the application of a primary antibody and a corresponding secondary antibody conjugated with horseradish peroxidase, specific for either mouse or rabbit immunoglobulins (Akoya Biosciences). Slides were then treated with various Opal fluorophores (1:100 dilution) in 1X Plus Amplification Diluent (Akoya Biosciences). After the tyramide signal amplification and the covalent linkage of individual Opal fluorophores to their respective epitopes, antibodies were removed by antigen retrieval, and the subsequent immunostaining cycle was started. All slides were counterstained with spectral DAPI (Akoya Biosciences) and mounted using Anti-fade fluorescence mounting medium (ab104135, Abcam). Multichannel imaging was executed using a PANNORAMIC SCAN II Imaging System (3Dhistech, Hungary) at × 200 magnification.

Vascular endothelial cell preparation and treatment

VECs were isolated from SD rats’ pulmonary veins. The rat was anesthetized, sterilized, and the thoracic cavity was opened to access the vein. Vascular tissues were cut into 1 mm squares, placed in a culture flask and incubated at 37 °C. Once attached, ECM medium (ScienCell, USA; containing 5% fetal bovine serum) was added. After three days, cells moving at the bottom were identified as VECs and those from passages 3–5 were used for further studies. To simulate a vascular endothelial injury model in vitro, LPS at a concentration of 1 μg/mL was added and allowed to incubate with the VECs for 24 h. To assess the protective effects of PTL, the VECs were pre-treated with 1 µM PTL 30 min before the LPS induction [7].

Observation of mitochondrial morphology

VECs were incubated with Mito-tracker (1:10,000) for 30 min at 37 °C and visualized using a Leica SP5 confocal microscope. Mitochondrial length was analyzed with a Mitochondrial Network Analysis (MiNA) toolset of Image J [7].

Detection of mitochondrial membrane potential and reactive oxygen species

To assess mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels, cells were seeded at 1 × 105 cells/mL in confocal dishes and incubated overnight at 37 ℃. Post-incubation, cells were rinsed with culture medium. JC-1 or 10 mM DCFH-DA solutions were added, mixed, and incubated for 15 min at 37 ℃ with CO2. ROS were measured at 488/525 nm using a Leica SP5 microscope, capturing red and green fluorescence at 590/610 nm and 485/535 nm, respectively. The red/green fluorescence ratio indicated mitochondrial membrane potential.

Mitochondrial oxygen consumption rate detection

The oxygen consumption rate (OCR) was measured using a 24-well XFe plate from Seahorse, Agilent, USA. VECs were seeded at 1 × 104 cells per well, incubated for 1 h, and then left overnight for adherence. Once 70–80% confluent, cells were treated with LPS and PTL for 24 h. They were prepared for 50 min in assay medium with 2.5 μM glucose and 2 mM glutamine, followed by sequential addition of 2 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone/antimycin A. The OCR was measured with an extracellular flux analyzer to induce mitochondrial stress.

Promoter activity assay (luciferase reporter gene system)

The reporter gene plasmid, transcription factor expression plasmid, and internal reference plasmid were constructed. Cells in their logarithmic growth phase were made into a cell suspension and inoculated into a 24-well culture plate (105 cells/ plate), then incubated in a 37 °C, 5% CO2 incubator until the cell confluence reached about 60%. The plasmids were transfected using X-tremegene HP (ROCHE) and dissolved in 100 µL of opti-MEM. The expression of the fluorescently labeled gene on the plasmid was observed 48 h after transfection to assess transfection efficiency. During the transfection of the target plasmid, an equal amount of GFP plasmid was transfected separately to demonstrate that the transfection system was functioning normally. A luciferase assay was conducted 48 h post-transfection to determine whether the transcription factor interacted with the promoter.

Chromatin immunoprecipitation (Chip)-qPCR detection

The cells were collected and fixed by cross-linking with 1% formaldehyde. The reaction was then terminated with glycine, and the cells were washed with PBS before the cell pellets were collected. During lysis and micrococcal nuclease treatment, the cells were processed to obtain chromatin fragments. Next, ChIP was performed, using a BRD4-specific antibody to bind the target chromatin, and the complex was captured with Protein A/G Agarose. After several washes, the DNA was eluted from the resin and purified using a DNA Clean-Up column. Finally, the purified DNA was quantified using qPCR to determine the enrichment level of the target regions. The BCL-xL promoter primer series can be found in Supplementary Table 1.

Preparation and characterization of N-PTL

The preparation of N-PTL used Kolliphor HS-15 as a surfactant, PEG-400 as a co-surfactant, and MCT in a 4:2:1 ratio. PTL was dissolved in MCT, then mixed with HS-15 and PEG-400, stirred at 300 rpm for 30 min at 25 °C. It was then left at room temperature for 30 min. The solution was diluted 100 times with double-distilled water and stirred until clear and slightly bluish. Droplet size and zeta potential were measured with a Zetasizer Nano ZS, morphology with a transmission electron microscope, and the infrared absorption spectrum was assessed.

Pharmacokinetic testing of N-PTL

The analysis used a Diamonsil C18 column (4.6 mm × 150 mm, 5 μm) with acetonitrile and water as the mobile phase in gradient elution. The flow rate was 1.0 mL/min, column temperature 30℃, and detection wavelength 210 nm. A 10 mg PTL standard was diluted in acetonitrile to create working solutions (200, 100, 50, 25, and 12.5 ng/mL) for a PTL standard curve. Blood was collected from rats at various times (0 min, 4 h, 12 h, 24 h, 48 h, 72 h). After centrifugation, supernatant was mixed with methanol (1:3), left for 30 min, and centrifuged at 12,000 rpm for 10 min at 4 ℃. Supernatant underwent nitrogen blow (1–2 L/min, 30–40 ℃) to obtain precipitate, which was re-dissolved in 0.1% acetonitrile. This process was repeated to obtain the supernatant.

Uptake percentage of PTL and N-PTL

PTL and N-PTL, each with fluorescein isothiocyanate (FITC) at 1 μmol/L, were added to cultured VECs in 96-well plates. After incubation for 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h, cells were rinsed with PBS, treated with trypsin, and their fluorescence measured at 485 nm using a microplate reader. Uptake was calculated as: Uptake (%) = (Z/Z0) × 100%, where ‘Z’ is the fluorescence of cell suspension at different times, and ‘Z0’ is the initial fluorescence with FITC.

Statistical analysis

Statistical analysis was conducted using SPSS version 20.0 and GraphPad Prism version 8.0. Data were presented as means ± standard deviation (SD). Each experiment involving animal data was independently replicated 6 times, while cellular data experiments were independently replicated at least 3 times. An independent sample t-test was utilized to analyze differences between two groups. For experiments with more than two groups, a one-way analysis of variance (ANOVA) was applied, followed by Tukey’s post hoc test and SNK/LSD comparisons. A P-value of less than 0.05 was considered statistically significant.

Results

PTL could alleviate SIC by improving VECs function

The results of HE staining showed significant changes in the lung histopathology of rats after sepsis, characterized by increased leukocyte infiltration, alveolar rupture, and pulmonary capillary congestion. PTL treatment can improve lung injury in septic rats (Fig. 1A). The level of tumor necrosis factor-α (TNF-α) in septic rats significantly increased and the survival rate significantly decreased. However, PTL treatment can effectively reduce TNF-α levels and increase the 48-h survival rate of septic rats (Fig. 1B, C). Furthermore, we observed the effect of PTL on the coagulation function of septic rats. The thrombelastograghy (TEG) results showed that septic rats had significant coagulation dysfunction, characterized by shortened activated coagulation time (ACT) time and decreased K value, increased Angle value and maximum amplitude (MA) value, significantly increased fibrinogen (FIB), and significantly shortened bleeding time, displaying a noticeable hypercoagulable state (Fig. 1D–J). However, PTL treatment can effectively improve the coagulation function of septic rats, characterized by increased ACT time and K value, decreased Angle value and MA value, significantly reduced FIB, and prolonged bleeding time (Fig. 1D–J). Due to the significant role of VECs in the regulation of coagulation function, we investigated the changes in pulmonary VECs following sepsis, as well as the effects of PTL on these cells, using immunohistochemistry. VECs were identified using the markers CD31 and VE-Cadherin. The results demonstrated that the number of VECs (CD31+/VE-Cadherin+) significantly decreased after sepsis. However, PTL was found to effectively mitigate VECs damage (Fig. 1K, L). Additionally, at the cellular level, we found that LPS stimulation can cause VECs dysfunction, characterized by increased expression of E-selectin, decreased expression of Thrombomodulin and VE-Cadherin, and decreased cell viability, whereas PTL treatment can improve VECs function (Fig. 1M, N).

Fig. 1
figure 1

PTL can be used to reduce SIC. A Observing the effect of PTL on sepsis-induced lung injury in rats using HE staining (n = 6 rats each group). B Measured TNF-α levels by Elisa (n = 6 rats each group). C The effect of PTL on the survival rate of septic rats after 48 h (n = 16 rats each group). TEG detected (D) ACT, (E) K value, (F) Angle value, and (G) MA value. H FIB level (n = 6 rats each group). The effect of PTL on (I) the percentage of rat bleeding and (J) tail bleeding time (n = 6 rats each group). K The change in the number of VECs detected by immunohistochemistry (bar = 40 μm) (n = 6 rats each group). L Quantification of CD31+/VE-Cadherin+ cells. M The effect of PTL on cell viability (n = 3 independent experiments). N WB was used to detect the impact of PTL on VECs function (n = 3 independent experiments). a: P < 0.05 as compared with the Sham or Normal group, b: P < 0.05 as compared with the Sepsis or LPS group

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PTL enhanced mitochondrial function and inhibited mitochondrial-mediated apoptosis in VECs

Our observations revealed that PTL has a protective effect on VECs after sepsis. We further conducted transcriptomic sequencing on normal and LPS-stimulated VECs, screened differential genes (|log2FoldChange|> 0, p-value < 0.05, LPS vs. Nor), and performed KEGG enrichment analysis. The results indicated that LPS stimulation could induce apoptosis in VECs (Fig. 2A). We treated LPS-stimulated VECs with various inhibitors of cell death or autophagy and assessed their viability. The results demonstrated that Emr (an apoptosis inhibitor), Nec-1 (a necroptosis inhibitor), and Fer-1 (a ferroptosis inhibitor) significantly enhanced VECs viability following LPS treatment. However, 3-MA (an autophagy inhibitor) did not exhibit a similar effect. Among the inhibitors, Emr had the most pronounced improvement in cell viability (Fig. 2B). TUNEL and Annexin V-FITC assay results revealed that PTL could inhibit LPS stimulation-induced VEC apoptosis (Fig. 2C, D). Moreover, we identified PTL’s influence on the mitochondrial structure and functionality within VECs. Our findings highlighted that mitochondrial fragmentation, hindrance in MMP, excessive ROS generation and decreased mitochondrial OCR, all caused by LPS stimulants, can be significantly rectified with PTL treatment (Fig. 2E–L). These results demonstrated PTL’s potential in mitigating mitochondrial-mediated apoptosis in VECs.

Fig. 2
figure 2

PTL enhanced mitochondrial function and inhibited mitochondrial-mediated apoptosis in VECs. A The KEGG enrichment analysis of differentially expressed genes in the transcriptome sequencing of VECs (LPS vs. Nor). B The effect of different inhibitors on the viability of LPS-treated cells was detected using a CCK8 assay (n = 3 independent experiments). C TUNEL was used to detect VECs apoptosis (bar = 25 μm) (n = 3 independent experiments). D Annexin V-FITC was used to detect VECs apoptosis (bar = 100 μm) (n = 3 independent experiments). E The mitochondrial morphology in VECs was observed using transmission electron microscopy (bar = 400 nm) (n = 3 independent experiments). F Confocal microscope observation of representative images of mitochondrial structure (bar = 8 μm) (n = 3 independent experiments). G The statistical analysis of mitochondrial morphology of VECs in different groups, each group randomly selected 10 cells and the mitochondrial morphology was blindly scored and classified into two categories: Long (> 3 µm), Short (≤ 3 µm). H, I JC-1 was used to detect the MMP (bar = 50 μm) (n = 3 independent experiments). J Effects of PTL on the mitochondrial OCR in VECs (n = 3 independent experiments). K, L DCFH-DA was used to detect the generation of ROS (bar = 100 μm) (n = 3 independent experiments). a: P < 0.05 as compared with the Normal group, b: P < 0.05 as compared with the LPS group

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PTL inhibited mitochondrial mediated apoptosis by up-regulating BCL-xL

To elucidate the mechanism by which PTL regulates the mitochondrial function of VECs, we first identified 100 potential target sites of PTL using network pharmacology, based on data from the Swiss Target Prediction database (Fig. 3A, B). We then collected normal and LPS-stimulated VECs for transcriptomics detection, followed by a differential analysis. We used venn analysis to intersect the differential genes, PTL’s target sites, and mitochondria-associated genes (we integrated four databases, including MitoCarta, MitoMiner, IMPI2, and UniProt to obtain a complete list of mitochondria-associated genes) [26]. The unique target B-cell lymphoma-extralarge (BCL-xL) was finally obtained, which was significantly down-regulated after LPS stimulation, whereas PTL could significantly up-regulate the expression of BCL-xL (Fig. 3C, D).

Fig. 3
figure 3

PTL inhibited mitochondrial mediated apoptosis by up-regulating BCL-xL. A PTL chemical structure. B Network pharmacology analysis of PTL target. C Venn diagram screens PTL action on mitochondrial-associated genes, PTL: PTL target, MITO: Mitochondrial-associated genes, UP: Up-regulation differential genes in LPS vs Nor, DOWN: Down-regulation differential genes in LPS vs Nor. D WB detection of PTL’s effect on BCL-xL expression (n = 3 independent experiments). E WB detection of the interference efficiency of shBCL-xL (n = 3 independent experiments). F, G Knocking down BCL-xL’s effect on mitochondrial morphological structure after being treated with PTL (bar = 10 μm), each group randomly selected 10 cells and the mitochondrial morphology was blindly scored and classified into two categories: Long (> 3 µm), Short (≤ 3 µm). H, I Knocking down BCL-xL’s effect on MMP after being treated with PTL (bar = 40 μm) (n = 3 independent experiments). J, K Knocking down BCL-xL’s effect on ROS after being treated with PTL (bar = 100 μm) (n = 3 independent experiments). L, M Knocking down BCL-xL’s effect on cell apoptosis, as detected by TUNEL (bar = 20 μm) and Annexin V (bar = 100 μm), after treatment with PTL (n = 3 independent experiments). N Knocking down BCL-xL’s effect on VECs function after being treated with PTL (n = 3 independent experiments). O WB detection of PTL’s effect on BCL-xL expression in vivo (n = 3 independent experiments). P WB detection of A-133 interference with BCL-xL (n = 3 independent experiments). Q The effect of BCL-xL inhibitor on the bleeding time of septic rats (n = 6 rats each group). R, S The effect of BCL-xL inhibitor on the change in the number of VECs (bar = 40 μm) (n = 6 rats each group). a: P < 0.05 as compared with the LPS + PTL or Sepsis + PTL group

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To determine whether PTL inhibits mitochondria-mediated apoptosis in VECs during sepsis by upregulating BCL-xL, we used adenoviral to knock down BCL-xL expression (Fig. 3E). Subsequently, we examined the effects on mitochondrial morphology and function. The results showed that compared with the PTL-treated group, the knockdown of BCL-xL could damage the mitochondrial morphology, leading to mitochondrial fragmentation, lowering MMP, and increasing ROS (Fig. 3F–K). Meanwhile, the knockdown of BCL-xL could impair VECs function and up-regulate VECs apoptosis (Fig. 3L–N). We further validated in an in vivo model using rat pulmonary vein tissues that PTL can upregulate BCL-xL, and used the specific BCL-xL inhibitor A-133 to downregulate BCL-xL (Fig. 3O–P), and found that inhibiting BCL-xL could counteract the protective effect of PTL on the coagulation function and VECs protective function in septic rats (Fig. 3Q–S).

PTL promoted the expression of BCL-xL by up-regulating BRD4

We further studied the mechanism of PTL upregulating BCL-xL. Utilizing the hTFtarget database (https://guolab.wchscu.cn/hTFtarget), we identified 9 transcription factors (TF) related to BCL-xL (Supplementary Table 2). After intersection with the target of PTL, we found that the BRD4 regulated BCL-xL with PTL (Fig. 4A). The molecular docking result showed that PTL can stably bind with BRD4, and its ASN-140 site can form a hydrogen bond with PTL (Fig. 4B). WB results showed that sepsis can down-regulate BRD4 levels, while PTL treatment can effectively up-regulate BRD4 levels (Fig. 4C, D). In addition, using adenovirus to knockdown BRD4 can effectively antagonize the upregulating effect of PTL on BCL-xL (Fig. 4E). The promoter activity was subsequently tested, and it was found that BRD4 has a positive regulatory effect on the BCL-xL promoter (Fig. 4F). The ChIP-qPCR results showed a significant enrichment of BRD4 at the five promoter primer sites of BCL-xL, and the enrichment was significantly reduced in the LPS-treated group compared to the normal group, indicating that LPS stimulation inhibits BCL-xL transcription through the downregulation of BRD4 (Fig. 4G). We further observed the effect of knocking down BRD4 on VECs apoptosis and mitochondrial function. The results demonstrated that the suppression of BRD4 will impair the function of VECs, increase apoptosis in VECs treated with PTL, significantly decreased mitochondrial OCR, which suggested that PTL enhanced BCL-xL by increasing BRD4, thereby influencing mitochondrial-mediated apoptosis (Fig. 4H–L).

Fig. 4
figure 4

PTL enhanced BCL-xL expression through up-regulation of BRD4. A The Venn diagram was used to screen the transcription factors that regulate BCL-xL expression by PTL. B PTL molecular docking with BRD4. C, D WB detection of the influence of PTL on BRD4 levels in vivo and vitro (n = 3 independent experiments). E Verification of the interference efficiency of adenovirus on BRD4 (n = 3 independent experiments). F The luciferase assay was used to detect promoter activity (n = 3 independent experiments). G The effects of LPS stimulation on BRD4 regulation of BCL-xL transcription were detected by Chip-qPCR (n = 3 independent experiments). H CCK8 detection of the impact of knocking down BRD4 on VECs apoptosis (n = 3 independent experiments). I, J Tunel (bar = 20 μm) and Annexin V-FITC (bar = 100 μm) detection of the impact of knocking down BRD4 on VECs apoptosis after being treated with PTL (n = 3 independent experiments). K The impact of knocking down BRD4 on VECs function (n = 3 independent experiments). L The impact of knocking down BRD4 on VECs mitochondrial OCR (n = 3 independent experiments). a: P < 0.05 as compared with the BCL-xL promoter-luc-NC + BRD4-NC group, b: P < 0.05 as compared with the BCL-xL promoter-luc-NC + BRD4 group, c: P < 0.05 as compared with the BCL-xL promoter-luc + BRD4-NC group, d: P < 0.05 as compared with the Normal group, e: P < 0.05 as compared with the LPS + PTL group

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The characterization detection of N-PTL

To enhance the protective effect of PTL, we utilized nanotechnology to construct N-PTL. The Mass Spectral results of N-PTL are 1H NMR (400 MHz, DMSO-d6) δ 6.12 (d, J = 3.7 Hz, 1H), 5.79 (d, J = 3.3 Hz, 1H), 5.25 (ddd, J = 12.3, 3.8, 1.8 Hz, 1H), 4.05 (dd, J = 9.0, 8.2 Hz, 1H), 3.01–2.88 (m, 2H), 2.44–2.29 (m, 1H), 2.22 (dd, J = 12.9, 6.0 Hz, 1H), 2.21–1.96 (m, 4H), 1.81–1.68 (m, 1H), 1.66 (d, J = 1.3 Hz, 3H), 1.20 (s, 3H), 1.18–1.05 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 169.76, 140.50, 135.03, 124.95, 121.12, 82.57, 65.70, 61.74, 46.91, 42.07, 40.98, 36.70, 36.38, 30.07, 24.16, 17.34, 17.09, which confirmed successful synthesis of N-PTL (Fig. 5A, B). The results depicted an average particle size of N-PTL being (106.7 ± 14.88) nm, and the Zeta potential was measured to be (− 17.0 ± 4.55) MV (Fig. 5C, D). In the PTL–COO– infrared spectrum, principal features were recorded at 1755 cm−1 and doublet peaks at 2979cm−1 and 2934cm−1 represented –CH3 in PTL whilst a broader peak at 3450 indicated –OH groups of PTL (Fig. 5E). The Electron Microscope image reveal that the N-PTL nano micelles are spherical, and showed regular particles (Fig. 5F). Moreover, the uptake rate of N-PTL was significantly higher than that of PTL from 2 h, suggesting that the nanotechnology was able to enhance the cellular uptake of PTL (Fig. 5G). Comparison between the pharmacokinetic curves of PTL and N-PTL indicated that N-PTL provided higher plasma concentration peak, reduction in apparent volume of distribution and clearance rate along with decreased elimination half-life. Furthermore, a significant increase was observed in area under the curve confirming that concentration of N-PTL in the blood outweighed PTL and exhibited increased residence time in the body (Fig. 5H).

Fig. 5
figure 5

The characterization detection of N-PTL. A LC–MS spectrum of N-PTL. B Nuclear magnetic resonance (NMR) hydrogen spectrum of N-PTL. C Zeta potential of N-PTL. D Size distribution of N-PTL. E Infrared spectroscopy of N-PTL. F Representative image of TEM of N-PTL (bar, 1 µm) (n = 3 independent experiments). G Uptake rates of PTL and N-PTL in VECs. H The pharmacokinetic curves of PTL and N-PTL (n = 3 independent experiments)

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N-PTL ameliorated SIC by inhibiting mitochondria-mediated apoptosis

The results show that compared to PTL, N-PTL has a stronger upregulation effect on BCL-xL and BRD4 in vivo and vitro (Fig. 6A–D). We further examined the improvement of N-PTL on the mitochondrial morphology and function of VECs, the inhibition apoptosis of VECs, and the impact on the coagulation function of septic rats. The results indicated that compared to PTL, N-PTL has superior protective effects on mitochondrial morphology and function, is more effective at reducing mitochondrial fragmentation, increases mitochondrial OCR and MMP, and inhibits ROS (Fig. 6E–K). In addition, compared to PTL, N-PTL can further inhibit VECs apoptosis, improve coagulation function, and reduce VECs damage in septic rats (Fig. 6L–R).

Fig. 6
figure 6

N-PTL suppressed mitochondrial-mediated apoptosis ameliorating SIC. A, B The effect of N-PTL on BCL-xL expression (n = 3 independent experiments). C, D The effect of N-PTL on BRD4 expression (n = 3 independent experiments). E The impact of N-PTL on VECs mitochondrial OCR (n = 3 independent experiments). F, G The influence of N-PTL on VECs mitochondrial morphological structure (bar = 8 µm). H, I The impact of N-PTL on VECs MMP (bar = 40 µm) (n = 3 independent experiments). J, K The effect of N-PTL on VECs ROS (bar = 100 µm) (n = 3 independent experiments). L Tunel detection of the influence of N-PTL on VECs apoptosis (bar = 20 µm) (n = 3 independent experiments). M Annexin V-FITC detection of the influence of N-PTL on VECs apoptosis (bar = 100 µm) (n = 3 independent experiments). N CCK8 detection of the effect of N-PTL on VECs apoptosis (n = 3 independent experiments). O, P The influence of N-PTL on the bleeding time in sepsis rats (n = 6 rats each group). Q, R The influence of N-PTL on the number change of VECs in sepsis rats (bar = 40 µm) (n = 6 rats each group). a: P < 0.05 as compared with the LPS + PTL or Sepsis + PTL group

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Discussion

The study demonstrated that sepsis significantly impaired coagulation and VECs function, whereas PTL effectively restored these functions. On a cellular level, VECs in sepsis exhibited apoptosis along with mitochondrial morphological and functional disruptions. PTL treatment, however, improved mitochondrial integrity and inhibited cell apoptosis. Mechanistic studies revealed that PTL enhanced the expression of BCL-xL by upregulating the transcription factor BRD4, thus inhibiting mitochondrial-mediated apoptosis in VECs and consequently improving coagulation function in sepsis. Furthermore, the use of nanotechnology to synthesize N-PTL further augmented the protective effects on VECs and coagulation function (Fig. 7).

Fig. 7
figure 7

Schematic diagram of this study. In sepsis, the downregulation of the transcription factor BRD4 in VECs leads to a decrease in BCL-xL levels, resulting in mitochondrial dysfunction and apoptosis, thereby causing coagulation disorders. PTL upregulates the expression of BCL-xL by enhancing BRD4 expression, inhibiting mitochondrial-mediated apoptosis in VECs, and ultimately ameliorating coagulation dysfunction in sepsis. Moreover, N-PTL, synthesized through nanotechnology, offers enhanced protective effects on VECs and coagulation function

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BCL-xL is an anti-apoptotic protein within the BCL-2 family, primarily located on the outer mitochondrial membrane, where it plays a key role in regulating the mitochondrial apoptosis pathway [27]. It forms heterodimers with pro-apoptotic BCL-2 family members such as Bax and Bak, thereby preventing these proteins from creating oligomeric pores on the mitochondrial membrane. This action inhibits the release of apoptotic signaling molecules like cytochrome C, consequently suppressing apoptosis [28]. Additionally, BH3-only proteins can compete with BCL-xL for binding, thereby relieving its inhibitory effect on Bax and Bak, which subsequently activates these proteins and induces apoptosis [29]. BCL-xL also impedes the binding of cytochrome C to Apaf-1, thus preventing the activation of Caspase-9 [30]. In VECs, BCL-xL plays a crucial role in apoptosis; studies have shown that inhibiting BCL-xL can lead to increased apoptosis of VECs infected with Kaposi’s sarcoma-associated herpesvirus (KSHV) [31]. Our study demonstrated that PTL can inhibit mitochondrial-mediated apoptosis in VECs by upregulating BCL-xL levels, thereby ameliorating SIC.

BRD4 is essential in regulating the transcription of numerous key genes involved in both physiological and pathological cellular processes, with a significant role in the regulation of the cell apoptosis pathway [32]. A distinctive feature of BRD4 is its two bromodomain structures, which bind acetylated lysine residues. Additionally, BRD4 interacts with positively charged amino acids to read and decode histones, influencing gene transcription [33, 34]. It is well-established that BRD4 inhibits cell apoptosis by regulating survival signals and suppressing apoptotic signals, including the BCL-2 family, p53, and NF-κB signaling pathways [35,36,37]. For instance, BRD4 enhances the transcriptional activation of the NF-κB signaling pathway by binding to the P-TEFb (positive transcription elongation factor b) complex, further inhibiting cell apoptosis [38]. This study demonstrated that PTL can increase BCL-xL levels by upregulating BRD4, thereby inhibiting VEC apoptosis and effectively treating sepsis-induced coagulopathy.

In recent years, nanotechnology’s growing significance in medicine has become increasingly evident. Notably, the nano-formulation of traditional Chinese medicine has garnered considerable interest due to its distinct advantages. Many active ingredients in traditional Chinese medicine exhibit limited water solubility, which adversely affects their bioavailability. However, these limitations can be addressed through the application of nanotechnology. By reducing the particle size and increasing the surface area, the process of nano-sizing medicinal constituents significantly improves solubility and enhances bioavailability [19, 39]. Moreover, nano-synthetic drugs show the potential to target lesions [40]. Finally, single-agent nanoformulations of traditional Chinese medicine offer notable advantages in the regulation of drug release. Nanotechnology modifies the drug release rate, facilitating the gradual release of the drug at a specific time and location. This prolongs the duration of drug action and optimizes therapeutic outcomes [41, 42]. In conclusion, nanoformulating traditional Chinese medicine enhances drug therapy effectiveness by improving solubility, precision targeting, and controlled release, ultimately leading to a reduction in side effects and an improved quality of life for patients.

This study has several limitations. While we found that PTL may exert protective effects by upregulating BCL-xL, we confined our verification to the animal level using a BCL-xL-specific inhibitor and did not employ endothelial cell BCL-xL conditional knockout mice. Additionally, the mechanisms by which PTL regulates BRD4 and the specific processes through which BRD4 influences BCL-xL transcription require further investigation.

Conclusions

This study elucidates the therapeutic effects and mechanisms of PTL on SIC, providing new strategies and directions for sepsis treatment.

Data availability

The raw data of this study are available from the corresponding author on reasonable request.

Abbreviations

5-HTR2A:

5-Hydroxytryptamine receptor 2A

ACT:

Activated coagulation time

BCL-xL:

B-cell lymphoma-extra large

BRD4:

Bromodomain-containing protein 4

CLP:

Cecal ligation and puncture

FIB:

Fibrinogen

FITC:

Fluorescein isothiocyanate

KSHV:

Kaposi’s sarcoma herpesvirus

LPS:

Lipopolysaccharide

MA:

Maximum amplitude

MMP:

Mitochondrial membrane potential

NF-κB:

Nuclear factor-kappa B

N-PTL:

Nano-PTL

OCR:

Oxygen consumption rate

P-TEFb:

Positive transcription elongation factor b

PTL:

Parthenolide

ROS:

Reactive oxygen species

SIC:

Sepsis-induced coagulopathy

TEG:

Thromboelastogram

TNF-α:

Tumor necrosis factor-α

VECs:

Vascular endothelial cells

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Acknowledgements

The authors would like to thank Jianyu Zhou from Chongqing Liyun Biotechnology Co., Ltd for his technical support.

Funding

This study was supported by the National Natural Science Foundation of China (No. 82300561 and 82305006), the Natural Science Foundation of Chongqing (No. CSTB2023NSCQ-MSX0713 and CSTB2022NSCQ-MSX0174), the Chongqing Talent Program: Innovative leading talents (No. CSTC2024YCJH-BGZXM0011), the Youth Program of Joint Medical Research of Chongqing Science and Chongqing Health Commission (No. 2025QNXM038), the Chongqing Natural Science Foundation Innovation and Development Joint Fund (Chongqing Education Commission) (No. CSTB2024NSCQ-LZX0079), and the Chongqing Medical Young Talents Program (No. YXQN202452 and YXQN202415).

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Author notes

  1. Jun Zhang, Xing Zhu and Yong Li contributed equally to this work.

Authors and Affiliations

  1. Department of Anesthesiology, Daping Hospital, Army Medical University, No.10, Changjiang Road, Yuzhong District, Chongqing, 400042, China

    Jun Zhang, Xing Zhu, Yong Li, Yinyu Wu, Yunxia Du, Hai Yang, Zhengchao Liu, Haoyu Pei, Rui Li, Han She & Qingxiang Mao

  2. Department of Critical Care Medicine, Chongqing General Hospital, Chongqing University, Chongqing, 401147, China

    Huan Luo

  3. Department of Rehabilitation Medicine, The First Affiliated Hospital of Chongqing University of Chinese Medicine, Chongqing Traditional Chinese Medicine Hospital, No.6, Panxi 7Th Branch Road, Jiangbei District, Chongqing, 400021, China

    Deyu Zuo

  4. Department of Research and Development, Chongqing Precision Medical Industry Technology Research Institute, Chongqing, 400000, China

    Deyu Zuo

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  1. Jun Zhang
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Contributions

Jun Zhang: Writing-original draft, Investigation, Formal analysis, Data curation, Conceptualization. Xing Zhu: Writing—original draft, Investigation, Formal analysis, Data curation. Yong Li: Investigation, Formal analysis, Data curation. Yinyu Wu: Supervision, Methodology. Yunxia Du: Formal analysis. Hai Yang: Data curation. Zhengchao Liu: Formal analysis. Haoyu Pei: Formal analysis. Rui Li: Data curation. Huan Luo: Data curation. Deyu Zuo: Supervision, Methodology, Conceptualization. Han She: Writing—review and editing, Supervision, Methodology, Conceptualization. Qingxiang Mao: Writing-review and editing, Supervision, Formal analysis, Conceptualization.

Corresponding authors

Correspondence to Deyu Zuo, Han She or Qingxiang Mao.

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Experiments involving animals were conducted in strict adherence to the guidelines provided by the Animal Research: Reporting of In Vivo Experiments (ARRIVE). The procedures were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (Approval No. AMUWEC20237118).

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Zhang, J., Zhu, X., Li, Y. et al. Parthenolide improves sepsis-induced coagulopathy by inhibiting mitochondrial-mediated apoptosis in vascular endothelial cells through BRD4/BCL-xL pathway. J Transl Med 23, 80 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06114-0

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Journal of Translational Medicine

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