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Journal of Traditional and Complementary Medicine logoLink to Journal of Traditional and Complementary Medicine
. 2024 Oct 28;15(7):726–735. doi: 10.1016/j.jtcme.2024.10.004

Parthenolide alleviates doxorubicin-induced cardiotoxicity via regulation of Cyp1a1 and Nppa and suppression of NLRP3 inflammasome

Yuanying Yang a,b, Shanshan Wei a,b, Sa Liu a,b, Suifen Xie a,b, Wei Xiao c, Jian Liu a,b, Ning Xie d, Wenqun Li a,b,⁎, Bikui Zhang a,b,⁎⁎
PMCID: PMC12624639  PMID: 41262674

Abstract

Background and aim

Doxorubicin (Dox) has limited clinical use due to its multiple adverse reactions, typically severe cardiotoxicity. Parthenolide (PTL) is the main active ingredient extracted from the buds of Tanacetum balsamita and exhibits diverse pharmacological properties. However, the cardioprotective effects and underlying mechanisms of PTL against Dox-induced cardiotoxicity (DIC) need to be fully investigated. Herein, this study was designed to explore the protective mechanism of PTL against DIC.

Experimental procedure

A stable cardiotoxicity model was established in H9c2 cells (1 μM for 24 h) and C57BL/6 J mice (15 mg/kg). RNA sequencing was used to identify key genes mediating the protection of PTL against DIC. The key genes and mechanism of PTL against DIC were comprehensively examined by transcriptomic technologies and experimental validation.

Results and conclusion

A combination administration of PTL effectively inhibited Dox-induced cytotoxicity as well as cardiomyocyte apoptosis in H9c2 cells. PTL also exerts a protective effect on Dox-induced cardiac injury by improving myocardial function, histological morphological changes, and myocardial apoptosis in Dox-treated mice. Subsequently, we utilized the transcriptomic approach and validated the results by RT-qPCR, confirming that Cyp1a1 and Nppa were the key genes in PTL against DIC. PTL could also protect from DIC via the suppression of the NLRP3 inflammasome activation and subsequent secretion of IL-1β and Caspase1. Our study confirmed that PTL treatment attenuated DIC in mice and H9c2 cells via regulation of Nppa and Cyp1a1 and the suppression of the NLRP3 inflammasome activation and subsequent secretion of pro-inflammatory cytokines.

Keywords: Parthenolide, Doxorubicin, Cardiotoxicity, RNA sequencing, NLRP3 inflammasome

Graphical abstract

Image 1

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Highlights

  • •

    Parthenolide protects against doxorubicin-induced cardiotoxicity.

  • •

    Nppa and Cyp1a1 mediate the protective effect of parthenolide on doxorubicin-induced cardiotoxicity.

  • •

    Parthenolide could alleviate Dox-induced cardiotoxicity by inhibiting the activation of NLRP3.

1. Introduction

Doxorubicin (Dox), an anthracycline antibiotic with a broad anti-cancer spectrum, is a first-line chemotherapeutic agent in clinical practice. However, Dox causes cytotoxic effects on normal cells in organs, especially on the heart, in a dose-dependent manner, often manifesting as left ventricular insufficiency, arrhythmias and even heart failure,1 which leads to a poor prognosis for cancer survivors. Therefore, the adverse effects of Dox, especially cardiotoxicity, have greatly hindered its use in clinical practice. The molecular mechanisms of DIC include inflammation, oxidative stress, apoptosis, mitochondrial damage, and dysregulation of autophagy.2 Despite the in-depth understanding of the mechanisms of DIC, the exact mechanisms are still not fully elucidated and the effectiveness of targeted therapy is limited. Attempts to develop chemical analogs of Dox that retain anticancer properties but reduce cardiotoxicity have met with little success.3 Clinically, only the iron ion chelator dexrazoxen has been approved by the U.S. Food and Drug Administration (FDA) for the prevention of DIC.4,5 Given the widespread use of Dox in chemotherapy, there is an urgent need to find novel agents and strategies to tackle DIC.

Parthenolide (PTL Fig. 1A), a natural sesquiterpene lactone originally extracted from the buds of Tanacetum balsamita, possesses anticancer, anti-inflammatory, antibacterial and other biological activities.6, 7, 8 PTL has also been regarded as a useful therapy for certain cardiovascular diseases. It has been reported that PTL can inhibit microtubule degradation and increase the contraction and relaxation rate of failing cardiomyocytes, which may be beneficial in the treatment of heart failure.8,9 Moreover, PTL blocked STAT3 signaling and alleviated angiotensin II-induced left ventricular hypertrophy by modulating fibroblast activity.10 However, there have been no studies on the effects of PTL on DIC. In our preliminary study, we observed a protective effect of PTL against DIC, but the mechanism through which PTL acts is not yet known.

Fig. 1.

Fig. 1

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PTL attenuated Dox-induced cardiac dysfunction in mice. (A) The chemical structure of PTL. (B) Schematic diagram of the animal dosage regimen. (C) Representative M-mode echocardiographs of left ventricular chamber and (D–G) the calculation of FS%, EF%, LVIDs and LVIDd. (H–K) Measurement of myocardial injury biomarkers including LDH (H), CK (I), CK-MB (J), and cTnT (K). Con: control; Dox: doxorubicin; PTL: Parthenolide; PTL-10: 10 mg/kg; PTL-20: 20 mg/kg; EF%: left ventricular ejection fraction; FS%: left ventricular fractional shortening; LVIDs: left ventricular end-systolic internal dimension; LVIDd: left ventricular end-diastolic internal; LDH: lactate dehydrogenase; CK: creatinine kinase; CK-MB: creatinine kinase-MB; cTnT: troponin T. Data are mean ± S.E.M., n = 6–8, ∗P < 0.05, ∗∗P < 0.01, vs Con; #P < 0.05, ##P < 0.01, vs Dox.

In this study, RNA sequencing (RNA-seq), a global transcriptomic approach, was used to analyze the gene expression differences in mice myocardial injury treatment by PTL and explore the possible targets of PTL in the treatment of DIC.

2. Material and methods

2.1. Cell culture

The H9c2 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium F12 (HyClone Co. Ltd, Logan, USA) supplemented with 10 % fetal bovine serum (FBS; Biological Industries Co. Ltd, Israel) and 1 % penicillin/treptomycin (Gibco Invitrogen, CA, USA) at 37 °C in a humidified incubator with 5 % CO2.

2.2. Cell viability assay

H9c2 cells were seeded onto 96-well plates at a density of 1 × 104 cells per well and incubated for 24 h. Cells were pretreated with PTL (Spring & Autumn Biological Engineering Co., Ltd., Nanjing, China) for 1 h, followed by treatment with Dox (1 μM) and PTL (0.5–5 μM) for additional 24 h. Next, 100 μl of medium containing 10 % Cell Counting Kit-8 (CCK-8) solution (New Cell & Molecular Biotech Co. Ltd, Suzhou, China) was added to each well and the absorbance was measured at 450 nm with a microplate reader (Tecan, Switzerland) after 1 h of incubation at 37 °C.

2.3. Flow cytometry analysis

FITC Annexin V Apoptosis Detection Kit was purchased from BD Biosciences (New York, USA). Cells were collected and washed twice with precooled phosphate buffered saline (PBS) and then resuspended in 1 × binding buffer. After being stained with FITC Annexin V and propidium iodide (PI), cells were gently vortexed and incubated for 15 min at room temperature (25 °C) in the dark. Samples were analyzed on the Cytek NL3000 (Cytek biosciences, California, USA) within 1 h.

2.4. Animals and treatments

The animal experiments were approved by the Medicine Animal Welfare Committee of Xiangya School of Medicine (CSU-2022-0262; Changsha, China). Eight-week-old C57BL/6 J mice (40 females, 20–25 g) were purchased from Laboratory Animal Center, Xiangya School of Medicine, Central South University (animal license: SYXK-2020-0019; Changsha, China). All animals were housed in a specific pathogen free barrier system under standard laboratory conditions of 50–60 % relative humidity, 20–24 °C, with a 12 h light-dark circle and were free access to water and food. All the procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

The forty mice were randomly and equally divided into four groups (n = 10): Con, Dox, Dox + PTL-10 and Dox + PTL-20. Mice in the Dox group were intraperitoneally administered 3 mg/kg Dox (Zhejiang Hisun Pharmaceutical Co. Ltd, China) every other day for 5 times (total dose: 15 mg/kg). Mice in the Dox + PTL-10 and Dox + PTL-20 groups were intragastrically injected with PTL (10 mg/kg/day or 20 mg/kg/day), respectively. The specific dosing regimen is shown in Fig. 1B.

One day after the last Dox injection, all mice were anesthetized with pentobarbital sodium and sacrificed to collect blood and cardiac tissue samples immediately.

2.5. Echocardiography

The mice were deeply anesthetized with isoflurane at a concentration of 2 % and fixed on a platform, with chest hairs removed. Echocardiography was performed using a Vevo 2100 ultrasound real-time imaging system (FUJIFLM Visualsonis, Canada) to obtain functional parameters in a blinded manner.

2.6. Biochemical analysis

The blood samples were centrifuged at 3000 rpm for 15 min and the supernatant was collected. The serum levels of cardiac injury markers including creatine kinase (CK), creatine kinase isoenzymes (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin T (cTnT) were measured with a 7600 automated biochemistry analyzer (HITACHI, Tokyo, Japan).

2.7. Histopathology analysis

4 % paraformaldehyde-fixed and paraffin-embedded cardiac tissue samples were prepared and cut into 4 μm slices, which were subjected to hematoxylin and eosin (H&E) staining. H&E staining was conducted to evaluate the degree of inflammatory cell infiltration and myocardial fibrosis. TdT-mediated dUTP Nick-End Labeling (TUNEL) staining was performed according to the standard protocol to assess apoptosis in cardiac tissues.11

2.8. RNA-seq analysis and data processing

Heart tissues were randomly collected from three groups (Con, Dox, and Dox + PTL) with 100–200 mg for 4 samples from each group for RNA-seq analysis. Total RNA was extracted with Trizol reagent (Thermofisher, Massachusetts, USA) according to the manufacturer's instructions. NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) was applied to assess the quantity and purity and Bioanalyzer 2100 (Agilent, CA, USA) was used to detect the integrity of RNA. The cDNA libraries were bipartite sequenced on the Illumina NovaseqTM 6000 (LC Biotechnology Ltd., Hangzhou, China). DESeq2 was applied to screen differential expressed genes (DEGs), with p-value <0.05 and |log2FC|> 0.585 as significance criteria. The Venn map was applied to screen the candidate DEGs, which were down-regulated in Dox group and up-regulated in Dox + PTL group, or DEGs up-regulated in Dox group and down-regulated in Dox + PTL group. The shared DEGs were regarded as the potential targets of PTL in the treatment of DIC. The DEGs were then subjected to GO functional enrichment and KEGG pathway analysis using the OmicStudio tools at https://www.omicstudio.cn/tool.

2.9. Immunohistochemical analysis

Paraffin-embedded cardiac samples were cut into 4 μm slices and then incubated with anti-NLRP3 antibody (1:300; Servicebio; Wuhan, China) or IL-1β (1:500; Abcam, Cambridge, UK) at 4 °C overnight. Next, the sections were incubated with secondary antibody for 30 min at room temperature. 3, 30-diaminobenzidine (DAB) chromogenic solution was added to the sections and then counterstained with hematoxylin to detect the expression of NLRP3 and IL-1β.

2.10. Western blotting

Protein lysates were collected from heart samples and H9c2 cells using enhanced RIPA Lysis Buffer containing 1 % protease inhibitor PMSF and phosphatase inhibitors (Boster Biological Technology Co. Ltd, Wuhan, China). The total protein concentrations of all samples were estimated using a BCA Protein Assay kit (Boster Biological Technology Co. Ltd, Wuhan, China). 20 μg total protein was separated on 10–12 % SDS-PAGE gels and transferred to 0.45 μm PVDF membranes (Millipore, Massachusetts, USA). Blocked with 5 % skim milk for 2 h, the membranes were incubated with anti-GAPDH (1:1000; ProteinTech, Wuhan, China), anti-Bcl2, anti-Caspase 3, anti-NLRP3, and anti-IL-1β (1:1000; Abcam, Cambridge, UK) at 4 °C overnight, respectively. Next, the membranes were incubated with HRP-conjugated affinipure goat anti-rabbit or anti-mouse IgGs (1:1000; ProteinTech, Wuhan, China).

Protein bands were determined by the ChemiDocTM imaging system and analyzed with Image LabTM software version 6.0 (Bio-Rad Laboratories Inc., Hercules, CA, USA).

2.11. Statistical analysis

Data values are depicted as mean ± standard error of means (SEM). Statistical analyses were performed using GraphPad Prism Version 7.00 Software (San Diego, CA, USA). One-way ANOVA followed by the Student–Newman–Keuls test was employed to conduct multiple comparisons. Statistical significance was defined when p < 0.05.

3. Results

3.1. PTL attenuated Dox-induced cardiac dysfunction in mice

Mice received Dox intraperitoneally (15 mg/kg) to establish DIC mice model. Animals in Dox + PTL-10 and Dox + PTL-20 groups were pretreated with PTL for one week and gavaged daily until the end of administration (Fig. 1B). Cardiac function was monitored via echocardiography before anatomy. There was a marked reduction in fraction shortening (FS) and ejection fraction (EF) after Dox treatment, while the left ventricular end-systolic internal dimension (LVIDs) and left ventricular end-diastolic internal (LVIDd) were significantly increased compared to the control group. Cotreatment with PTL markedly rescued reduction in FS and EF and reversed the increase in LVIDs and LVIDd as compared with the Dox group in a dose-dependent manner (Fig. 1C–G). Dox-induced increases in the serum levels of cardiac injury markers including CK-MB, CK, LDH and cTnT were reversed by PTL (Fig. 1H–K). Thus, these results above demonstrate that PTL could protect from Dox-induced cardiac dysfunction.

3.2. PTL suppressed Dox-induced cardiomyocyte apoptosis in mice

Histological analysis showed Dox-induced structural disorders in myocardial tissues, which were significantly relieved by PTL pre-treatment (Fig. 2A). TUNEL staining was employed to examine the degree of cardiomyocyte apoptosis. We noticed a significant decrease in the number of TUNEL-positive cells in mice pretreated with PTL (Fig. 2B). Changes in the expression of apoptosis-associated proteins, such as pro-apoptotic protein Bax and anti-apoptotic protein Bcl2, can also indicate apoptosis. PTL increases the ratio of Bcl-2 to Bax, which was decreased by Dox stimulation, indicating that PTL could ameliorate Dox-induced apoptosis (Fig. 2C-D).

Fig. 2.

Fig. 2

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PTL suppressed Dox-induced cardiomyocyte apoptosis in mice. (A) Representative images of H&E staining in the myocardium. (B) Representative images of TUNEL staining. (C–D) Protein level of the Bcl-2/Bax ratio in cardiac tissues of mice, as measured by western blotting. Con: control; Dox: doxorubicin; PTL: parthenolide; PTL-10: 10 mg/kg; PTL-20: 20 mg/kg. Data are mean ± S.E.M., n = 6, ∗P < 0.05, ∗∗P < 0.01, vs Con; #P < 0.05, ##P < 0.01, vs Dox.

3.3. PTL attenuated Dox-induced cardiomyocyte apoptosis in H9c2 cells

Subsequently, we further investigated the role of PTL on Dox-induced cardiomyocyte apoptosis in H9c2 cells. CCK-8 assay was conducted after treating cells with different concentrations of Dox for 24 h. Based on the results above, 1 μM Dox was chosen to establish a stable cardiotoxicity model in subsequent experiments. The group with PTL treatment did not experience any cytotoxicity, but significantly ameliorated Dox-induced cell death, as evidenced by cell viability result (Fig. 3A). Flow cytometry analysis showed that PTL effectively inhibited the rate of Dox-induced apoptosis in cardiomyocytes. (Fig. 3B–C). Moreover, co-administration of PTL reversed the downregulation of the ratio of Bcl2/Bax that was generated by Dox, which was in accordance with in vivo results (Fig. 3D–E). Overall, these findings supported that PTL attenuated Dox-mediated cardiac apoptosis.

Fig. 3.

Fig. 3

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PTL attenuated Dox-induced cardiomyocyte apoptosis in H9c2 cells. (A) Cell viability of H9c2 cells pretreated with PTL (0.5–5 μM) for 1 h and then treated with both Dox and PTL for 48 h. (B–C) Apoptosis rate was determined by flow cytometry for H9c2 cells pretreated with PTL for 1 h and then treated with Dox and PTL for 48 h. (D–E) Protein level of the Bcl-2/Bax ratio in H9c2 cells. Con: control; Dox: doxorubicin; PTL: parthenolide; PTL-0.5: 0.5 μM; PTL-1: 1 μM. Data are mean ± S.E.M., n = 5. ∗P < 0.05, ∗∗P < 0.01 vs. Con; #P < 0.05, ##P < 0.01 vs. Dox.

3.4. mRNA profile and the identification of DEGs

In order to investigate the protective mechanism of PTL against DIC, RNA-seq was conducted to obtain DEGs in the Con group, the Dox group and the Dox + PTL group. The correlation heatmap results indicated that the samples were reasonable and the RNA-seq data were reliable (Fig. 4A). Furthermore, Principal component analysis (PCA) suggested good reproducibility of the samples within each group, with the PTL+Dox group and Con group distinct from Dox group (Fig. 4B). Using P < 0.05, |log2FC|>0.585 as the screening condition, a total of 5568 genes were up-regulated and 1415 genes were down-regulated by Dox treatment compared with the control group; There were 263 down-regulated genes and 333 up-regulated genes in the Dox + PTL group compared with the Dox group. Of all the genes whose expression changed after Dox treatment, 267 genes had their expression changes reversed by PTL, as shown in Fig. 4C. There were 105 genes remaining after removing genes with low gene expression (FPKM<1) and high heterogeneity, and the top 105 genes were visually represented in a heat map (Fig. 4D).

Fig. 4.

Fig. 4

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The analysis of RNA-seq. (A) Correlation heat map between pairs based on expression abundance. The redder the color, the higher the correlation is. (B) PCA diagram. (C) The Venn map of DEGs shows 157 genes downregulated by Dox and upregulated by PTL, and 110 genes upregulated by Dox and downregulated by PTL. (D) Cluster heat map of the top 100 DEGs with the largest |log2FC| values. A change in color from blue to red notes the expression level of the gene from low to high. (E–F) GO (E) and KEGG (F) analysis of the top 100 DEGs. The data for each group were obtained from six mice.

3.5. GO and KEGG pathway analysis of DEGs changed by PTL

In order to uncover the biological processes and pathways in which these 105 genes may be involved in the treatment of PTL against DIC, GO and KEGG analyses were performed using the omicstudio platform. GO pathway enrichment results showed that 105 DEGs of PTL were involved in 25 biological processes including oxidation-reduction, metabolic, regulation of transcription, DNA-templated, protein phosphorylation. In terms of cellular component, the nucleus, membrane, neclusome, and integral component of membrane was significantly enriched. In addition, the top 105 DEGs were related to DNA binding, protein binding, protein heterodimerization, and other molecular functions (Fig. 4E). KEGG analysis revealed multiple enriched biosynthesis and metabolism pathways (Fig. 4F).

3.6. RT-qPCR validation for DEGs in RNA-seq

To validate the RNA-seq results, we selected six DEGs associated with heart diseases (Cyp1a1, Nppa, Rnf144b, Timp4, Ucp3 and Pdk4) among the top 105 DEGs. We then detect the mRNA expression of these six DEGs in cardiac tissue by RT-qPCR. Among the six DEGs, only the RT-qPCR results of Cyp1a1 and Nppa were consistent with the RNA-seq data (Fig. 5A–L). The results showed that compared with the Con group, Dox increased the RNA levels of Cyp1a1 and Nppa, while PTL treatment significantly reversed the Dox-induced mRNA expression of these two DEGs.

Fig. 5.

Fig. 5

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RT-qPCR verification for RNA-seq. (A–F) Quantitative mRNA expression of Cyp1a1, Nppa, Rnf144b, Timp4, Ucp3 and Pdk4 in RNA-seq analysis. (G–L) The mRNA level of Cyp1a1, Nppa, Rnf144b, Timp4, Ucp3 and Pdk4 detected by RT-qPCR. Con: control; Dox: doxorubicin; PTL: parthenolide; PTL-20: 20 mg/kg. Data are mean ± S.E.M., n = 6–8, ∗P < 0.05, ∗∗P < 0.01, vs Con; #P < 0.05, ##P < 0.01, vs Dox.

3.7. PTL can inhibit NLRP3 inflammasome-mediated inflammation in Dox-treated mice and H9c2 cells

Myocardial levels of NLRP3, IL-1β and Caspase1 in mice were evaluated to determine the effect of PTL on NLRP3-induced inflammation. The expressions of NLRP3 and IL-1β were examined by IHC analysis. The increases in the expression of NLRP3 and IL-1β were reversed by PTL (Fig. 6A–B). As shown in Fig. 6C–G, the expression of NLRP3 and its downstream IL-1β, Caspase1 were upregulated after Dox administration, while co-administration with PTL led to a decrease in NLRP3, IL-1β and Caspase1. As expected, changes in IL-1β and NLRP3 were consistent with IHC results, further corroborating the above findings. Collectively, these results demonstrated that PTL plays a part in inhibiting NLRP3 inflammasome-mediated inflammation induced by Dox. We further evaluated the effect of PTL on NLRP3-mediated inflammation in H9c2 cells. Western blotting results indicated that NLRP3, IL-1β tended to upregulate after Dox treatment compared to control group in vitro; while PTL cotreatment significantly decreased the expression of NLRP3, IL-1β in a dose-dependent manner (Fig. 6H–J). Overall, these findings unveiled that PTL can protect from inflammation mediated by NLRP3 inflammasome in Dox-treated mice and cardiomyocytes.

Fig. 6.

Fig. 6

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PTL can inhibit NLRP3 inflammasome-mediated inflammation in Dox-treated mice. (A–B) The IL-1β (A) and NLRP3 (B) immunohistochemistry in cardiac tissues. (C–D) The representative results of NLRP3, Casepase 1, pro IL-1β and cleaved IL-1β expression in cardiac tissues calculated by western blotting. (E–F) Quantification of results from (C–D). (H) The representative results of NLRP3, pro IL-1β and cleaved IL-1β expression in H9c2 cells measured by western blotting. (I–J) Quantification of results from (H). Con: control; Dox: doxorubicin; PTL: parthenolide; PTL-10: 10 mg/kg; PTL-20: 20 mg/kg; PTL-0.5: 0.5 μM; PTL-1: 1 μM. Data are mean ± S.E.M., n = 6–8 (mice) or n = 5 (H9c2 cell), ∗P < 0.05, ∗∗P < 0.01 vs. Con; #P < 0.05, ##P < 0.01 vs. Dox.

4. Discussion

Dox is a first-line chemotherapeutic agent for the treatment of solid tumors. However, its adverse effects, especially cardiotoxicity, greatly limit its clinical use. Dexrazoxane is the only FDA-approved drug for the prevention and treatment of DIC, but although it has been demonstrated that dexrazoxane does not affect the antitumor effect of anthracyclines, its risk of triggering second malignant neoplasm is controversial.12 Scientists have turned their attention to chemical compounds derived from natural products with a wide range of pharmacological activities and low toxicity in the hope of finding effective drugs to counteract DIC. Parthenolide, a naturally occurring substance of the plant Tanacetum parthenium, displays various beneficial effects, including anti-tumor, anti-viral, anti-inflammatory and anti-atherosclerosis properties.8,13, 14, 15 Several studies have shown that PTL administration improves cardiovascular disease. PTL exerts beneficial effects on cardiovascular dysregulation and prognosis in endotoxic shock and attenuates myocardial reperfusion injury.16,17 Previous studies revealed that Dox-induced cardiomyocyte injury is mainly mediated by DNA damage and apoptosis,18 which suggested that anti-apoptotic therapy may exert beneficial effects on cardiac injury.19 To assess Dox-induced apoptosis, we measured the degree of apoptosis using TUNEL staining and flow cytometry analysis and detected Bcl-2/Bax ratio in the cardiac tissue of Dox-treated mice. We found that Dox activated the apoptotic pathway, demonstrated by the increase of apoptotic cells and the decrease in the Bcl-2/Bax ratio. Interestingly, PTL cotreatment significantly increased the Bcl-2/Bax ratio and reduced the number of apoptotic cells, which revealed that PTL attenuated Dox-induced cardiomyocyte apoptosis. In our study, we provide evidence for the protection of PTL against DIC in vivo and in vitro for the first time and explore the mechanisms with the help of RNA-seq.

Cyp1a1 encodes CYP1A1, a member of the cytochrome P450 (CYP450) enzyme superfamily. CYP1A1 metabolizes arachidonic acid into the cardiotoxic product 20-HETE, which plays an important role in the development of cardiac hypertrophy and heart failure.20 Early reports showed that CYP1A1 mRNA expression was detected in both the right ventricle and left atrium of patients with dilated cardiomyopathy.21 Induced expression of the Cyp1a1 gene was also observed in isoproterenol-induced cardiac hypertrophy in rats.22 In our study we found that Cyp1a1 mRNA levels were increased after DIC in mice, which is consistent with the study performed by Volkova.23 A combination administration of PTL inhibited the increase in Cyp1a1 mRNA levels caused by Dox, which is the first report that PTL regulates the expression of Cyp1a1 gene.

Nppa gene is mainly expressed in the heart, with higher expression levels in the atria than in the ventricles, and is responsible for encoding the precursor of atrial natriuretic polypeptide (ANP).24 As a cardiac hormone, ANP can act as a diuretic, natriuretic and vasodilator, thereby regulating water-salt balance and blood pressure. Moreover, ANP has essential multiple effects on the cardiovascular system. Studies have shown that ANP has antihypertrophic functions in the heart, but not its systemic blood pressure lowering effects.25 In Nppa knockout mice, dietary or drug treatment lowered blood pressure but did not prevent cardiac hypertrophy.26 Furthermore, the Nppa gene was significantly upregulated in isoproterenol-induced cardiac hypertrophy and fibrosis,27 consistent with the changes in mice with diabetic cardiomyopathy.28 Nppa expression is reactivated in the ventricles during hypertrophy and heart failure, which is thought to be part of a highly conserved adaptive change in gene expression.29 In our study, Nppa expression was induced to upregulate after Dox administration, which was reversed by PTL.

The discovery by Emad S. Alnemri that PTL is the first potent natural inhibitor of NLRP3 inflammasomes which directly target NLRP3 and rapidly inhibits caspase-1 activation and IL-1β maturation at low concentrations, has attracted our attention.30 Our previous study revealed that activation of the NLRP3 inflammasome and consequent secretion of pro-inflammatory cytokines give play to the occurrence of DIC.31 Here, we showed that Dox activated NLRP3 inflammasome, as evidenced by upregulation of NLRP3, Caspase-1 and IL-1β. It has been reported that specific inhibitors of NLRP3, such as MCC-950 and oridonin, attenuated DIC.32,33 Therefore, drugs that target NLRP3 inflammasome may be prospective candidates for protection against Dox-induced cardiac injury. We sought to investigate whether the protective effect of PTL against DIC might be exerted by inhibiting NLRP3. In our present study, we found that PTL attenuated DIC by inhibiting NLRP3 and thereby reducing IL-1β secretion and Caspase1 activation, as shown in the result of IHC analysis and western blotting. However, we did not detect any changes in NLRP3 mRNA levels in the transcriptomic results, which may be due to the fact that PTL directly targets NLRP3 protein without affecting the transcription of its mRNA.

This study systematically elucidated the potential mechanism of PTL against DIC by transcriptomic techniques. It was speculated that PTL may protect from DIC by regulating the expressions of Cyp1a1 and Nppa and inhibiting the NLRP3 inflammasome activation. Unfortunately, our study still has some limitations. Firstly, we only verified the expression changes in mRNA levels of Cyp1a1 and Nppa without further validation at the protein levels. Additionally, our future studies should carry out further experimental verification of NLRP3 as a mediator of cardioprotective effects in PTL, such as gene interference. Looking ahead, there are several promising avenues for further exploration. Many natural products have been shown to inhibit tumorigenesis and progression, and are important potential alternatives to chemotherapeutic drugs.34, 35, 36 Among these, PTL exerts powerful anti-tumor effects by inducing autophagy of tumor cells, inhibiting invasion and migration of tumor cells and other pathways.8,14,37 The dysregulation of autophagy promotes carcinogenesis and the progression of cancer, rendering this process an indispensable target for cancer therapy.38 It has been reported that autophagy degraded components of the NLRP3 inflammasome in a p62-dependent manner.39 Additionally, autophagosomes targeted IL-1β in macrophages following TLR activation.40 Therefore, whether PTL can increase the antitumor effect of Dox by inducing autophagy and inhibiting NLRP3 while attenuating DIC is unknown and remains to be demonstrated experimentally. Despite PTL has made progress in preventing DIC in our study, the main hindrance to converted it into clinical use is its poor water solubility and low bioavailability. Therefore, strategies such as structural modification and changing dosage forms are necessary. The antitumor activity of PTL has now been greatly improved by structural modification and changing dosage,41, 42, 43 but whether these approaches can improve the ability to protect against DIC remains to be further explored.

5. Conclusion

In summary, our study confirmed that PTL treatment attenuated DIC in mice and H9c2 cells via regulation of Nppa and Cyp1a1 and the suppression of the NLRP3 inflammasome activation and subsequent secretion of pro-inflammatory cytokines. PTL could be developed as a novel therapeutic candidate for the prevention of DIC.

Section

Natural Products.

Taxonomy (classification by EVISE)

Identify the disease/health condition.

Funding

This study was supported by grants of the National Natural Scientific Foundation of China (Nos. 82173911, 82373970, 81973406), Fundamental Research Funds for the Central Universities of Central South University (Nos. 2023ZZTS0556), Hunan Provincial Natural Scientific Foundation (Nos. 2020JJ4823, 2022JJ80109), Hunan Provincial Health Commission scientific research project (No, D202313017647), Scientific Research Project of Hunan Provincial Health and Family Planning Commission (No. 202113050843), Research Project established by Chinese Pharmaceutical Association Hospital Pharmacy department (No. CPA-Z05-ZC-2021-002), Changsha Science and Technology Project (kq2004137), and Chinese Anti- Cancer Association HER2 target Chinese Research Fund (CORP-239-S5).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Peer review under responsibility of The Center for Food and Biomolecules, National Taiwan University.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jtcme.2024.10.004.

Contributor Information

Wenqun Li, Email: liwq1204@csu.edu.cn.

Bikui Zhang, Email: 505995@csu.edu.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

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