Leonurine inhibits cardiomyocyte pyroptosis to attenuate cardiac fibrosis via the TGF-β/Smad2 signalling pathway

Zhaoyi Li; Keyuan Chen; Yi Zhun Zhu

doi:10.1371/journal.pone.0275258

. 2022 Nov 3;17(11):e0275258. doi: 10.1371/journal.pone.0275258

Leonurine inhibits cardiomyocyte pyroptosis to attenuate cardiac fibrosis via the TGF-β/Smad2 signalling pathway

Zhaoyi Li ^1,^2,^#, Keyuan Chen ^1,^2,^#, Yi Zhun Zhu ^1,^2,^*

Editor: Luis Eduardo M Quintas³

PMCID: PMC9632889 PMID: 36327230

Abstract

Cardiac fibrosis is a common cause of most cardiovascular diseases. Leonurine, an alkaloid from Herba leonuri, had been indicated to treat cardiovascular diseases due to its cardioprotective effects. Recently, pyroptosis, a programmed form of cell death that releases inflammatory factors, has been shown to play an important role in cardiovascular diseases, especially cardiac fibrosis. This study examined the novel mechanism by which leonurine protects against cardiac fibrosis. In rats with isoprenaline-induced cardiac fibrosis, leonurine inhibited the expression of proteins related to pyroptosis and improved cardiac fibrosis. In vitro, leonurine inhibited the expression of proteins related to pyroptosis and fibrosis. Additionally, leonurine regulated the TGF-β/Smad2 signalling pathway and inhibited pyroptosis to protect cardiomyocytes and improve cardiac fibrosis. Therefore, leonurine might improve cardiac fibrosis induced by isoprenaline by inhibiting pyroptosis via the TGF-β/Smad2 signalling pathway.

Introduction

Cardiac fibrosis, which is also called myocardial calcification, is an irreversible process that causes a stiff heart and influences cardiac function, such as cardiac relaxation and contraction, leading to many cardiovascular diseases, such as heart failure, myocardial infarction, atherosclerosis, myocardial hypertrophy and other coronary heart diseases [1]. Certain stimulating factors, including transforming growth factor-β (TGF-β), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β), trigger the signal transduction pathway to promote cardiomyocytes to differentiate into myofibroblasts, resulting in a large amount of fibrin production and extracellular matrix deposition. Eventually, this process leads to cardiac fibrosis [2, 3]. Pyroptosis is one of the causes of various cardiovascular diseases [4, 5]. Pyroptosis is a new type of programmed cell death caused by cysteinyl aspartate specific proteinase 1 (caspase 1) or caspase 11 [5]. Morphologically, pyroptosis has features of apoptosis and necrosis. Unlike apoptosis, pyroptosis is usually accompanied by the formation of pores, causing membrane rupture and cell death, especially the release of inflammatory factors [6–8]. The caspase 1-dependent pathway is the canonical pyroptosis pathway [9]. After inflammasome activation, activated caspase 1 and cleaves gasdermin D (GSDMD), which forms cell membrane pores that promote the maturation and release of proinflammatory cytokines (IL-1β and IL-18), ultimately leading to pyroptosis [4, 10]. According to literature, the NOD-like receptor protein 3 (NLRP3) inflammasome increases collagen synthesis and the expression of IL-1, IL-18, and caspase 1 in myocardial fibroblasts [11, 12], all of which are essential in the pathogenesis of cardiac fibrosis. In addition, Caspase 1 activation, GSDMD cleavage and cell membrane perforation promote the secretion of IL-1 and IL-18, which exacerbate myocardial ischaemia–reperfusion injury [13]. Leonurine, which is an alkaloid in Herba leonuri, can treat myocardial infarction [14], which is an acute cardiac disease [15], and attenuates cardiac fibrosis after myocardial infarction by inhibiting NADPH oxidase 4 [16]. However, there has been no report on leonurine as a treatment for heart failure, which is a chronic heart disease [17]. Therefore, in this study, the cardioprotective effect of leonurine against heart failure in rats was investigated in the heart failure rat model induced by isoprenaline (ISO), and the effect of leonurine on cardiac fibrosis was examined in a TGF-β-induced cardiac fibrosis model in vitro. We showed for the first time that leonurine could treat cardiac fibrosis by inhibiting pyroptosis.

Materials and methods

Materials

ISO was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). CMC-Na was purchased from Macklin (Shanghai, China). Leonurine was synthesized by Professor Zhu Yi Zhun’s lab at Fudan University according to the reported method [14] and was kindly provided. Lactate dehydrogenase (LDH) and creatine kinase (CK) kits were obtained from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). A detergent-compatible Bradford protein assay kit (Beyotime, Nantong, China) was used.

Animal experiments

A total of 30 male Sprague–Dawley rats (4–6 weeks old, 220–250 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China) (licence: SCXK (Beijing) 2019–0010). The rats were randomly divided into five groups, including the control group (n = 6), the model group (n = 6), and three leonurine groups (n = 6 per group) that were administered low (25 mg/kg/day), medium (50 mg/kg/day), and high (100 mg/kg/day) doses. The control group was subcutaneously injected with normal saline for 48 consecutive days. All rats, except for those in control group, were subcutaneously injected with ISO to establish a heart failure as previously reported [18]. A total of 5 mg/kg ISO was administered on the first day, and 2.5 mg/kg ISO was administered for the remaining 47 consecutive days. The rats in the three leonurine groups received low-, medium-, and high-dose leonurine orally for 48 consecutive days after ISO induction. No rats died during this experiment. At the end of the 48-day treatment period, the rats were anaesthetized with 2% pentobarbital (0.2 mL/100 g, i.p.), blood samples were collected from the abdominal aorta in vacutainers containing sodium heparin. Blood samples were immediately centrifuged at 4500 ×g for 15 min, and plasma samples were collected and stored at -80°C for later use. The rats were sacrificed by cervical dislocation, and the hearts were quickly removed and washed with ice-cold phosphate buffered saline and stored at -80°C for later use.

All animal procedures were conducted according to the Guidelines for the Care and Use of Laboratory Animals (Version 8) and were approved by the Animal Ethics Committee of the Animal Center of the State Key Laboratory of Quality Research of Traditional Chinese Medicine, Macau University of Science and Technology. All surgeries were performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering.

Haemodynamic measurement

At the end of the eight-week treatment, the rats were anaesthetized with 2% pentobarbitone (0.3 ml/200 g, i.p.). A metal signal detector was inserted into the right carotid artery to record the left ventricular systolic pressure (LVSP), left ventricular end-diastole pressure (LVEDP), the rising rate (+dP/dt max), and the falling rate (-dP/dt max) (ADI MPVS-Ultra Single Segment Foundation System, BME_036, Macau University of Science and Technology, Macau, China).

Biochemical analysis

Plasma levels of LDH and CK were measured using detection kits (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China), and plasma levels of IL-1β were measured using a detection kit (MULTI SCIENCES, Hangzhou, China) according to the manufacturer’s instructions.

Histology analysis

The heart tissues were embedded in paraffin and fixed in 4% paraformaldehyde for at least twenty-four hours. The samples were then cut into five mm pieces and set on slides according to a routine procedure. The heart sections were stained with Masson’s trichrome, haematoxylin and eosin (H&E), and Sirius red (PSR), and ImageJ software was used for analysis (National Institutes of Health, Bethesda, MD, USA).

Cell culture and treatments

The H9c2 rat embryonic ventricular myocardial cell line was purchased from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium (containing 10% foetal bovine serum and 1% penicillin–streptomycin) at 37°C in 95% air/5% CO₂. H9c2 cells were subjected to 20 ng/mL TGF-β for 30 minutes or 48 hours. H9c2 cells were pretreated with 20 μM leonurine for 4 hours before TGF-β stimulation.

Western blot analysis

Protein was extracted from cultured H9c2 cells and rat heart tissue. The protein concentration was determined by a detergent-compatible Bradford protein assay kit (Beyotime, Nantong, China). Protein samples were subjected to 8% and 12% SDS–PAGE and blotted to a nitrocellulose filter membrane. The blots were blocked with 5% BSA, followed by incubation with the indicated primary antibodies, including mouse anti-β-actin, rabbit anti-Smad2, rabbit anti-phospho Smad2, rabbit anti-Gasdermin D, rabbit anti-cleaved Gasdermin D, rabbit anti-Caspase 1, rabbit anti-cleaved Caspase 1 (1:1000, Cell Signalling Technology), rabbit anti-α-SMA, mouse anti-COL3A1 and mouse anti-FN1 (1:500, Santa), overnight at 4°C. The membranes were then washed three times with TBST and incubated with secondary antibodies, including HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (diluted 1:10,000, ICL Lab, USA), for 2 hours at room temperature. The membranes were washed three times with TBST, and ECL western blotting detection reagent (Millipore) was used to develop the immunoblots. Protein bands were visualized by a GE Amersham Imager 800 RGB (EG, USA).

Immunofluorescence staining

H9c2 cells (3×10⁴ cells/well) were cultured on confocal dishes. After the different treatments, the cells were fixed in 4% paraformaldehyde for 10 minutes, followed by permeabilization with 0.3% Triton X-100 in PBS for 5 minutes. The cells were blocked with 5% BSA in PBS for 30 minutes at room temperature and incubated with Smad2 (1:1000, Cell Signalling Technology) antibodies at 4°C for 16 hours. Fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG (H+L) secondary antibodies (1:200, 134325, Jackson ImmunoResearch) were added and incubated for 2 hours at room temperature. The cells were counterstained with DAPI (Beyotime, Nantong, China), and images were captured by a fluorescence microscope (IX73, Olympus).

Measurement of ROS generation

The generation of intracellular reactive oxygen species (ROS) was examined using a Reactive Oxygen Species Assay Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Briefly, H9c2 cells (2×10⁴ cells/well) were grown in 6-well plates and subjected to different treatments. The cells were collected and centrifuged at 2000 rpm/min for 5 minutes, and the supernatant was discarded. Then, the H9c2 cells were incubated with 5 μM 2,7-dichlorofluorescein diacetate (DCFH-DA) at 37°C for 20 minutes, washed three times with PBS, and resuspended in the flow tubes in 500 μL of PBS. After being resuspended, the cells were analysed by using a flow cytometer (BD FACSAria III, USA).

Statistical analysis

The data are expressed as the mean ± standard error of the mean (mean ± SEM). All data analysis was performed with GraphPad Prism 8.0 software (GraphPad-Prism Software Inc., San Diego, CA, USA). Differences between the mean values of multiple groups were analysed by one-way analysis of variance with Tukey’s test for post hoc comparisons. Statistical significance was considered at P<0.05.

Results

Leonurine improved cardiac fibrosis and exerted cardioprotective effects in vivo

To examine the effects of leonurine on ISO-induced cardiac fibrosis, ISO-induced rats were treated with leonurine. After ISO induction, LVEDP, +dP/dt max, and -dP/dt max increased 1.62- (P<0.01), 2.36- (P<0.01), and 2.06-fold (P<0.01), respectively, and LVSP decreased 1.75-fold (P<0.01), which indicated that rat cardiac function worsened after ISO induction (Fig 1). In addition, LDH and CK increased 1.48- (P<0.01) and 1.41-fold (P<0.01), respectively, indicating that ISO caused myocardial tissue damage (Fig 2). High-dose (100 mg/kg/day) leonurine decreased the LVEDP, +dP/dt max, and -dP/dt max by 1.33- (P<0.05), 1.62- (P<0.05), and 1.81-fold (P<0.05), respectively, but low-dose and middle-dose leonurine could not significantly improve the cardiac function of rats with ISO-induced heart failure. Low, medium, and high doses of leonurine increased LVSP by 1.43 (P<0.05), 1.65 (P<0.01), and 1.74 times (P<0.01), respectively, which suggested that leonurine enhanced cardiac function in rats with ISO-induced heart failure (Fig 1). Moreover, low, medium, and high doses of leonurine decreased the levels of LDH by 0.85- (P<0.05), 0.68- (P<0.01) and 0.81-fold (P<0.05) and CK by 0.34- (P<0.001), 0.26- (P<0.001) and 0.49-fold (P<0.001), respectively (Fig 2). These results showed that leonurine could protect cardiac tissues from damage in rats with ISO-induced heart failure.

Fig 1 — Cardiac function is responsive to haemodynamic variables, including LVSP, LVEDP, -dP/dt max, and +dP/dt max. Leonurine increased (A) LVSP level, and decreased levels of (B) LVEDP, (C) -dP/dt max, and (D) +dP/dt max compared with the model group. ##P<0.01, control group vs. model group; **P<0.01, *P<0.05, leonurine groups vs. model group. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; -dP/dt max, the falling rate of LVEDP; +dP/dt max, the rising rate of LVEDP.

Fig 2 — Myocardial injury is responsive to LDH and CK. Leonurine decreased levels of (A) CK and (B) LDH in plasma in heart failure rats induced by ISO. #P<0.05, ##P<0.01, model group vs. control group; *P<0.05, **P<0.01, ***P<0.001, leonurine groups vs. model group. CK, creatine kinase; LDH, lactic dehydrogenase.

Cardiac fibrosis was serious after ISO induction, as evidenced by the area of cardiac fibrosis shown by H&E, Masson, and PSR staining (Fig 3), and the expression of proteins related to fibrosis, including α-SMA, COL3A1 and FN1, increased 2.19- (P<0.01), 3.97- (P<0.001), and 5.78-fold (P<0.001), respectively (Fig 4). Leonurine reduced the area of cardiac fibrosis, as evidenced by the staining results (Fig 3). Furthermore, leonurine inhibited the expression of α-SMA, COL3A1, and FN1 by 2.17- (P<0.01), 1.88- (P<0.01), and 2.10-fold (P<0.0.1) in the medium-dose group, respectively, and 2.24- (P<0.01), 3.36- (P<0.01), and 2.68-fold (P<0.01) in the high-dose group, respectively (Fig 4). These results suggested that leonurine enhanced cardiac function and improved cardiac fibrosis in rats with ISO-induced heart failure.

Fig 4 — (A) Compared to ISO-induced rats, leonurine decreased the expression of proteins related to fibrosis including (B) FN1, (C) α-SMA, and (D) COL3A1. ##P<0.01, ###P<0.001, model group vs. control group; **P<0.01, leonurine groups vs. model group.

Leonurine inhibited pyroptosis in vivo

Furthermore, in rats with ISO-induced heart failure, pyroptosis and inflammatory responses were exacerbated, including increases in GSDMD, cleaved GSDMD, caspase 1, cleaved caspase 1, and IL-1β by 1.56- (P<0.05), 2.87- (P<0.001), 2.14- (P<0.01), 2.67- (P<0.01), and 7.11-fold (P<0.001), respectively. However, high-dose (100 mg/kg/day) leonurine decreased the expression of proteins related to pyroptosis, including GSDMD, cleaved GSDMD, caspase 1, and cleaved caspase 1, by 1.03- (P<0.05), 1.57- (P<0.01), 1.15- (P<0.05), and 1.56-fold (P<0.01), respectively. Moreover, low, medium, and high doses of leonurine reduced the levels of IL-1β by 1.46- (P<0.05), 3.19- (P<0.001), and 4.49-fold (P<0.001), respectively, in a concentration-dependent manner, indicating that leonurine could inhibit pyroptosis after ISO induction in vivo (Fig 5).

Fig 5 — (A) Compared to ISO-induced rats, leonurine decreased expressions of pyroptosis proteins including (B) GSDMD, (C) cleaved GSDMD, (D) Caspase 1, (E) cleaved Caspase 1, and the level of (F) IL-1β in plasma. #P<0.05, ##P<0.01, ###P<0.001, model group vs. control group; *P<0.05, **P<0.01, ***P<0.001, leonurine groups vs. model group.

Leonurine inhibited pyroptosis in vitro

In vitro, leonurine reduced the expression of proteins related to fibrosis after TGF-β treatment, including α-SMA, COL3A1, and FN1, by 1.43- (P<0.01), 2.14- (P<0.001), and 1.39-fold (P<0.05), respectively (Fig 6), and these results were consistent with the animal experiment results. Moreover, leonurine downregulated the expression of cleaved GSDMD and cleaved caspase 1 by 1.38- (P<0.05) and 1.38-fold (P<0.05), respectively, and decreased the level of IL-1β by 1.52-fold (P<0.05) in TGF-β-induced H9c2 cells, indicating that leonurine could inhibit pyroptosis after TGF-β stimulation in vitro (Fig 7). Thus, we concluded that leonurine inhibited pyroptosis to attenuate cardiac fibrosis.

Fig 6 — (A) Compared to H9c2 cells induced by TGF-β for 48 hours, leonurine decreased the expression of proteins related to fibrosis including (B) FN1, (C) α-SMA, and (D) COL3A1. #P<0.01, ###P<0.001, model group vs. control group; *P<0.05, **P<0.01, ***P<0.001, 20 μM leonurine group vs. model group.

Fig 7 — (A) Compared to H9c2 cells induced by TGF-β for 48 hours, leonurine decreased the expression of proteins related to pyroptosis including (B) GSDMD, (C) cleaved GSDMD, (D) Caspase 1, (E) cleaved Caspase 1, and the level of (F) IL-1β. #P<0.05, model group vs. control group; *P<0.05, 20 μM leonurine group vs. model group.

Leonurine inhibited cardiomyocyte pyroptosis via the TGF-β/Smad2 signalling pathway in vitro

To gain an in-depth understanding of the role of leonurine in pyroptosis during cardiac fibrosis, we analysed the effects of the TGF-β/Smad2 signalling pathway on pyroptosis induced by cardiac fibrosis. After TGF-β stimulation, the levels of ROS and phosphorylated Smad2 were increased 2.13- (P<0.01) and 6.42-fold (P<0.001), respectively, indicating that the TGF-β/Smad2 signalling pathway was activated by TGF-β (Figs 8 and 9). Leonurine reduced the levels of ROS, phosphorylated Smad2, and cleaved caspase 1 by 2.11- (P<0.01), 6.23- (P<0.001), and 1.49-fold (P<0.05), respectively (Fig 9). These results suggested that leonurine regulated the TGF-β/Smad2 signalling pathway to inhibit cardiomyocyte pyroptosis.

Fig 8 — DCFH-DA was used to evaluate the intracellular ROS level. DCFH-DA is a nonfluorescent ester of the dye fluorescent product in the presence of ROS. (A) The peak value of the model group shifted to the right, and the peak value of the leonurine group shifted to the left. (B) ROS production increased with TGF-β exposure, and pretreatment with leonurine inhibited the ROS increase after TGF-β stimulation. ##P<0.01, model group vs. control group; **P<0.01, 20 μM leonurine group vs. model group. DCFH-DA, 2,7-dichlorofluorescein diacetate.

Fig 9 — (A) Compared to H9c2 cells induced by TGF-β for thirty minutes, leonurine decreased the expressions of Smad2 phosphorylation and cleaved Caspase 1 (B) The ratio of Smad2 phosphorylation to Smad2 expression and the ratio of cleaved Caspase 1 to Caspase 1 expression. (C) The fluorescence of Smad2 protein was detected under a confocal laser scanning microscope. Compare to TGF-β induced model group, the intensity fluorescence of Smad2 protein was weakened after leonurine treatment. #P<0.05, ##P<0.01, model group vs. control group; *P<0.05, ***P<0.001, 20 μM leonurine group vs. model group.

Discussion

Heart failure, which is a global epidemic with increasing morbidity and mortality each year, has become a global public health burden [19].

A novel finding of this study was that leonurine enhanced cardiac function in rats with ISO-induced heart failure by modulating the levels of haemodynamic variables, including LVEDP, LVSP, -dP/dt max, and +dP/dt max, and decreased the levels of LDH and CK, which reduced cardiomyocyte damage and injury. Furthermore, leonurine reduced the levels of collagen and fibres to alleviate cardiac fibrosis in rats with ISO-induced heart failure. ISO, which is a catechol substance, activates the sympathetic nervous system by nonselectively exciting β receptors on cardiomyocytes, causing the production and release of catecholamines in tissue cells and increasing the concentrations of catecholamines and vasoconstrictors in the blood circulation, which increases the heart rate [20]. Furthermore, leonurine could also reduce haemodynamic parameters and the levels of LDH and CK in rats with myocardial infarction induced by coronary artery ligation [21] and heart ischaemia induced by ligation of the left coronary artery [22], thereby exerting cardioprotective effects similar to those in this study. Therefore, we hypothesize that the cardioprotective mechanism of leonurine may be independent of β-adrenergic receptors.

When β-adrenergic receptors are overactivated by ISO in vivo, membrane nanotubes promote inflammasome activation to spread from cardiomyocytes to cardiac fibroblasts, resulting in cardiac fibroblast pyroptosis [23]. In vitro, TGF-β activated the NLRP3 inflammasome in cardiac fibroblasts, forming a pathogenic cycle that led to the development of cardiac fibrosis [24]. Interestingly, we found that leonurine attenuated the expression of pyroptosis proteins, including Caspase 1, cleaved Caspase 1, GSDME, and cleaved GSDME, in vivo and in vitro, improving cardiac function and protecting against cardiac fibrosis, as evidenced by reductions in fibrosis areas and collagen expression. Thus, we hypothesis that pyroptosis also plays an indelible role in the development of cardiac fibrosis and that leonurine might improve cardiac fibrosis by inhibiting pyroptosis.

Reportedly, the activation of Caspase 1 promoted the release of inflammatory factors such as IL-1β [25]. Moreover, IL-1β could promote fibrosis by regulating the TGF-1β/Smad2/3 pathway [26], which is a classic pathway associated with cardiac fibrosis that plays a key role in the differentiation of cardiac fibroblasts into myocardial fibroblasts and collagen deposition [27, 28]. TGF-β1 affects R-Smad proteins, activating Smad2/3 phosphorylation, promoting profibrotic gene expression and leading to cardiac fibrosis [29]. Our study showed that leonurine could inhibit Smad2 phosphorylation in a TGF-β-stimulated cardiac fibrosis cell model, demonstrating that leonurine was involved in the regulation of the TGF-β/Smad2 signalling pathway. Furthermore, leonurine reduced ROS production in the TGF-β-stimulated cardiac fibrosis cell model. It has been reported that ROS can not only activate apoptosis [30] but also mediate fibrotic responses [31], and the results suggest that leonurine may regulate TGF-β-induced cardiac fibrosis by inhibiting apoptosis by reducing ROS production. In addition, a previous study also obtained similar results showing that leonurine could regulate the TGF-β/Smad3 signalling pathway by inhibiting Smad3 phosphorylation to treat tubulointerstitial fibrosis in unilateral urethral obstruction (UUO) mice, and there were reductions in collagen Ⅰ/Ⅲ, ROS production and proinflammatory factors such as IL-1β, IL-6, and TGF-β [32]. Thus, based on these findings, we hypothesize that leonurine inhibits the activation of Caspase 1 and ROS production, preventing GSDMD cleavage and pore formation, thereby reducing the release of inflammatory factors such as IL-1β and attenuating the inflammatory response. After the activation of Caspase 1 was inhibited by leonurine, the release of inflammatory factors and Smad2 phosphorylation were reduced, which further inhibited TGF-β/Smad2 signalling pathway conduction and myofibroblast differentiation and reduced the expression of α-SMA, COL3A1, and FN1, thereby improving cardiac fibrosis.

In conclusion, our study was the first to show that leonurine enhanced cardiac function and improved cardiac fibrosis by decreasing the expression of pyroptosis proteins and fibrosis proteins in vivo and in vitro. In addition, leonurine might suppress cardiac fibrosis by inhibiting pyroptosis via the TGF-β/Smad2 signalling pathway (Fig 10). Although this study did not validate this mechanism by using inhibitors of the TGF-β/Smad2 signalling pathway or pyroptosis in vitro, we provide a basis for broadening the use of leonurine in the treatment of cardiac fibrosis.

Fig 10 — Leonurine inhibited the activation of Smad2 phosphorylation and regulated the TGF-β/Smad2 signalling pathway, thereby the expression of proteins related to pyroptosis including Caspase 1 and GSDMD. The release of a large number of inflammatory factors like IL-1β caused cardiac fibrosis and promoted the pathological process of heart failure. TGF-β, transforming growth factor-β; TGF R1, transforming growth factor receptor 1; TGF R2, transforming growth factor receptor 2; Smad2, small mothers against decapentaplegic 2; P-Smad2, phospho-Smad2; IL-1β, Interleukin 1 beta; Caspase 1, cysteinyl aspartate specific proteinase 1; GSDMD, gasdermin-D.

Supporting information

S1 Table. Hemodynamic variables.

(XLSX)

Click here for additional data file.^{(10.6KB, xlsx)}

S2 Table. OD values of CK, LDH, and IL-1β.

(XLSX)

Click here for additional data file.^{(12.4KB, xlsx)}

S3 Table. Western blot analysis.

(XLSX)

Click here for additional data file.^{(13.4KB, xlsx)}

S4 Table. ROS fluorescence intensity.

(XLSX)

Click here for additional data file.^{(9.8KB, xlsx)}

S1 Raw images. Original entire blots for figures.

(PDF)

Click here for additional data file.^{(458.2KB, pdf)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by the Macau Science and Technology Development Fund (FDCT 0007/2019/AKP, 0021/2020/AGJ, 0011/2020/A1). The National Natural Science Foundation of China (Nos. 81973320).

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PLoS One. doi: 10.1371/journal.pone.0275258.r001

Decision Letter 0

Luis Eduardo M Quintas

6 Jun 2022

PONE-D-22-05865Leonurine inhibits cardiomyocyte pyroptosis against cardiac fibrosis via the TGF-β/Smad2 signaling pathwayPLOS ONE

Dear Dr. Zhu,

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Reviewer #1: Yes

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**********

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5. Review Comments to the Author

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Reviewer #1: Dear author, your article is unpublished and has relevance in the field of study. However, it is necessary to pay attention to some questions that I put below for clarification:

Introduction

Page 9

“35 … After

36 respective inflammasomes activation, activated Caspase 1 and Cleaved

37 gasdermin D (GSDMD) to formed cell membrane pores that promote the

38 maturation and release of pro-inflammatory cytokines (IL-1β and IL-18),

39 ultimately leading to pyroptosis [4,10]...”

Sugested:

After

36 respectively inflammasomes activation, activated Caspase 1 and Cleaved

37 gasdermin D (GSDMD) to form cell membrane pores that promote the

38 maturation and release of pro-inflammatory cytokines (IL-1β and IL-18),

39 ultimately leading to pyroptosis [4,10].

Pags. 9 and 10

“39…Reportedly, in myocardial

40 fibroblasts, NOD-like receptor protein 40 3 (NLRP3) inflammasome

41 increased collagen synthesis and expressions of IL-1β, IL-18 and caspase

42 1 [11,12] which are playing an indispensable role in the pathogenesis of

43 cardiac fibrosis.”

Sugested:

“39…Reportedly, in myocardial

40 fibroblasts, NOD-like receptor protein 40 3 (NLRP3) inflammasome

41 increased collagen synthesis and expressions of IL-1β, IL-18 and caspase

42 1 [11,12], which plays an indispensable role in the pathogenesis of

43 cardiac fibrosis.”

Pags 9 and 10

“ 39…Reportedly, in myocardial

40 fibroblasts, NOD-like receptor protein 3 (NLRP3) inflammasome

41 increased collagen synthesis and expressions of IL-1β, IL-18 and caspase

42 1 [11,12] which are playing an indispensable role in the pathogenesis of

43 cardiac fibrosis. In additional, Caspase 1 activation, GSDMD cleavage and

44 cell membrane perforation and promoting secretion of IL-1β and IL-18,

45 which aggravated myocardial ischemia-reperfusion injury [13].

The text is confused. I suggest be more clear. Like:

“…According to reports, the NOD-like receptor protein 3 (NLRP3) inflammasome increased collagen synthesis and the expression of IL-1, IL-18, and caspase 1 in myocardial fibroblasts [11,12], all of which are essential in the pathogenesis of cardiac fibrosis. In addition, Caspase 1 activation, GSDMD cleavage and cell membrane perforation, and promoting secretion of IL-1 and IL-18, which aggravated myocardial ischemia-reperfusion injury…”

Pag. 10

“ …Therefore, present

55 study aims to examine whether leonurine improves cardiac fibrosis and its

56 possible mechanism in pyroptosis.”

Suggested:

“…Therefore, the present

55 study aims to examine whether leonurine improves cardiac fibrosis and its

56 possible mechanism in inhibition of pyroptosis.”

Materials and methods

Pag. 13

“ 117 followed by probed with the indicated primary antibodies including mouse…”

Sugested :

… followed by incubation with the indicated primary antibodies including mouse…”

Pag. 13

“120… rabbit anti-Smad2 phosphorylation

121 (1:1000, Cell Signaling),...”

Suggested:

“ 120… rabbit anti- phospho Smad2

121 (1:1000, Cell Signaling),…”

Pag. 14

“137…and incubated with Smad2 (1:1000, Cell Signaling)

138 antibodies at 4 ℃.”

For how long?

The experimental design is not entirely clear. When does Leonurine begin to be applied to the animal?

Another issue concerns the in vitro study. There is no reference to cell culture and treatment. It is necessary to describe the culture conditions, medium, culture time. And the experimental treatment design.

Results

Pag. 16

“164 After ISO treatment, rats were administered leonurine for eight weeks.

165 Levels of left ventricular end-diastolic pressure (LVEDP), its rising and

166 falling rate (±dP/dt max), lactic dehydrogenase (LDH) and creatine kinase

167 (CK) were significantly increased 1.53-, 2.01-, 2.31-, 1.48- and 1.41-fold,

168 and left ventricular systolic pressure (LVSP) was decreased 1.71-fold in

169 rats after ISO treatment (Fig 1 and 2).”

I suggest rewriting this section. Lots of information about the measured parameters. It could put the numerical data in an ordering closer to parameter groups. Ex: left ventricular end-diastolic pressure (LVEDP), its rising and falling rate (±dP/dt max) increased as shown respectively 1.53-, 2.01- and 2.31.

Pag. 17

“183 After leonurine treatment, expressions of 183 proteins related to

184 pyroptosis were decreased in the heart from rats induced by ISO (Fig 5).”

Sugested:

183... After leonurine treatment, the expression of proteins related to

According to the figures, it is clear that there are different answers in relation to the concentrations of leonurine used. These differences are not addressed during the discussion and there is no information for the reader to choose the concentration used in the cell culture treatment. The descriptor text of the results does not even indicate when there is a change in the use of animal tissue for cell culture. Clarity in writing is necessary for the understanding of the article to be total.

Figures:

Fig. 3 Areas of cardiac fibrosis in ISO-induced rats with leonurine treatment for eight weeks. Representative pictures of left ventricles from each group after H&E staining, Masson staining, and PSR staining (magnification, ×4, ×40). Leonurine decreased areas of collagen and fibers compared to the model group. (A) Collagen and fibers areas are pink after H&E staining. (B) Collagen and fibers areas are blue after Masson staining. (C) Collagen and fibers areas are yellow after PSR staining.

The description of figure A is interchanged with that of figure B.

In figure 8 there is an error in the first line of image A. It should be cleaved Capase 1.

Considerations:

I suggest that a rereading of the article be done in order to make the writing clearer.

The authors speak of the effect of leonurine in blocking the TGFbeta/SMAD-2 signaling pathway. An experiment in which leonurine was used and compared to a pathway inhibitor would be great.

Reviewer #2: Leonurine inhibits cardiomyocyte pyroptosis against cardiac fibrosis via the TGF-β/Smad2 signaling pathway

Reviewer Recommendation and Comments

I have had the pleasure of reading Li and colleagues’ manuscript. The research manuscript aimed to answer the question whether the alkaloid Leonurine can block the progression of cardiac fibrosis and elucidate the molecular mechanism involving pyroptosis. I would like to suggest an English review by a native English-speaking reviewer for improve the understanding of the reader. Moreover, I further have a few comments on specific points in other to improve the manuscript understanding.

Major Concern:

1. Title: “Leonurine inhibits cardiomyocyte pyroptosis against cardiac fibrosis via the TGF-β/Smad2 signaling pathway.” I think the word “against” leads to the reader a misunderstanding about the main view of the manuscript. I would suggest, “Leonurine inhibits cardiomyocyte pyroptosis via the TGF-β/Smad2 signaling pathway to prevent/block (the right word depends on the answer of question 4.c.) cardiac fibrosis.

2. Abstract: The citation (line 7): “The manuscript studied the novel mechanism…” please change for “This report…”.

3. Introduction: It is missing the working hypothesis of the work. It is confusing from line 51 through the end of the paragraph. Reference 16 mentioned in the manuscript is also about the attenuation of myocardial fibrosis. Moreover, I don´t understand what the authors means that it has a great cardioprotective effect. Are the authors related this sentence with cardiac function (LEVDP, LVSP, Dp/Dt) preservation? If the answer is yes, what is the new point of this manuscript? Please clarify the working hypothesis enhancing the differences from previous publications.

4. Materials and Methods:

a. Leuronine was obtained from Fudan University; to me it seems that the substance is not commercially available. Is it as extraction? The authors should provide more information.

b. The authors should provide the total n number and mention if the treatment induced any loss of the animals.

c. Treatment protocol is confusing. To all rats, ISO was injected during 48 days. When exactly Leuronine was orally administrated? During ISO administration or after? For how long, 8 days? This may change the interpretation of the data. If the Leuronine was treated after ISO administration for 8 days, the substance reverts cardiac fibrosis. This issue should be discussed.

5. Results: The quality of Fig 3 is poor. The yellow mentioned in the text can´t be seen.

6. Discussion:

a. Paragraphs 1 and 2 says the same things, please be concise.

b. ISO is a beta-adrenergic agonist. Do the ISO-induced pyroposis mediated by beta-adrenergic receptor or is a consequence of the sustained augmentation of cardiac haemodinamic that may stimulate others mediators? If the pyroposis is mediated beta-adrenergic receptor, what is the difference of the treatments with beta adrenergic antagonist (such as atenolol) and Leuronine. Are co-treatment with both drugs synergic? The authors should provide the data or discuss this issue.

c. Leuronine is an alkaloid, if ISO was administered in concomitance to Leuronine, the physically interaction of both drugs is possible? Or, is Leuronine able to bind to beta adrenergic receptor? Another possibility is ROS chelation? These points as missed in the discussion.

d. At the end of the discussion, the authors mentioned “some limitations”, the authors should cite and discuss it better.

Minor Concern:

1. Please, provide the p values of each result in order for a better conclusion of the reader.

2. Please, revise Figure legends 1, 2, 3 and 4. The letters that label’s the panels are exchanged.´

3. It is encourage that the authors draw a picture underling the proposed molecular mechanism of Leuronide.

Reviewer #3: The authors studied the novel mechanism of leonurine on cardiac fibrosis with its cardioprotective effects. They used both in vivo animal model and experiments on H9c2 cells were conducted. The manuscript is well written. The topic is interesting, and the results presented enhancing our existing knowledge. Even though the findings are important the authors must improve the manuscript. In this regard, the results section must be rewritten. It is not clear the experiments performed and if the results described were in the animal model or in the cells. In addition, the English needs to be improved.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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Attachment

Submitted filename: Review PONE-D-22-05865 LSLM.pdf

Click here for additional data file.^{(274.2KB, pdf)}

PLoS One. 2022 Nov 3;17(11):e0275258. doi: 10.1371/journal.pone.0275258.r002

Author response to Decision Letter 0

27 Jul 2022

Dear editor and reviewers,

Thank you for the opportunity to revise our manuscript PONE-D-22-05865 entitled “Leonurine inhibits cardiomyocyte pyroptosis against cardiac fibrosis via the TGF-β/Smad2 signaling pathway”. We are grateful for the detailed comments and suggestions provided by the reviewers, and those input are all greatly valuable and helpful for revising and improving our paper, as well as the important guiding significance to our researches.

Please kindly see below our response to the reviewers’ comments as well as our revised manuscript. We hope we have addressed the reviewers’ concerns satisfactorily and our manuscript is now fit for publication in PLOS ONE.

All best regards

Yours sincerely

Yi Zhun Zhu

Corresponding Author

Reviewer #1: Dear author, your article is unpublished and has relevance in the field of study. However, it is necessary to pay attention to some questions that I put below for clarification:

1. Introduction

a. Page 9, line 35-39. “After respective inflammasomes activation, activated Caspase 1 and Cleaved gasdermin D (GSDMD) to formed cell membrane pores that promote the maturation and release of pro-inflammatory cytokines (IL-1β and IL-18), ultimately leading to pyroptosis [4,10]...” Suggested: “After respectively inflammasomes activation, activated Caspase 1 and Cleaved gasdermin D (GSDMD) to form cell membrane pores that promote the maturation and release of pro-inflammatory cytokines (IL-1β and IL-18), ultimately leading to pyroptosis [4,10].”

Response: Thank you for the great suggestion. We have revised the descriptions accordingly in the Introduction section following your suggestion (Page 3, lines 37-41).

b. Pages 9 and 10, line 39-43. “Reportedly, in myocardial fibroblasts, NOD-like receptor protein 40 3 (NLRP3) inflammasome increased collagen synthesis and expressions of IL-1β, IL-18 and caspase 1 [11,12] which are playing an indispensable role in the pathogenesis of cardiac fibrosis.” Suggested: “Reportedly, in myocardial fibroblasts, NOD-like receptor protein 40 3 (NLRP3) inflammasome increased collagen synthesis and expressions of IL-1β, IL-18 and caspase 1 [11,12], which plays an indispensable role in the pathogenesis of cardiac fibrosis.”

Response: Thank you for the kind suggestion. We have revised the descriptions accordingly in the Introduction section following your suggestion (Page 4, lines 41-44).

c. Pages 9 and 10, line 39-45. “Reportedly, in myocardial fibroblasts, NOD-like receptor protein 3 (NLRP3) inflammasome increased collagen synthesis and expressions of IL-1β, IL-18 and caspase 1 [11,12] which are playing an indispensable role in the pathogenesis of cardiac fibrosis. In additional, Caspase 1 activation, GSDMD cleavage and cell membrane perforation and promoting secretion of IL-1β and IL-18, which aggravated myocardial ischemia-reperfusion injury [13].” The text is confused. I suggest be more clear. Like: “According to reports, the NOD-like receptor protein 3 (NLRP3) inflammasome increased collagen synthesis and the expression of IL-1, IL-18, and caspase 1 in myocardial fibroblasts [11,12], all of which are essential in the pathogenesis of cardiac fibrosis. In addition, Caspase 1 activation, GSDMD cleavage and cell membrane perforation, and promoting secretion of IL-1 and IL-18, which aggravated myocardial ischemia-reperfusion injury…”

Response: Thank you so much for your kind reminder. We have revised the descriptions accordingly in the Introduction section following your suggestion (Page 4, lines 41-47).

d. Page 10, line 55-56. “Therefore, present study aims to examine whether leonurine improves cardiac fibrosis and its possible mechanism in pyroptosis.” Suggested: “Therefore, the present study aims to examine whether leonurine improves cardiac fibrosis and its possible mechanism in inhibition of pyroptosis.”

Response: Thank you for the kind suggestion. We have revised the descriptions accordingly in the Introduction section following your suggestion (Page 4, lines 52-57).

2. Materials and methods

a. Page 13, line 117. “followed by probed with the indicated primary antibodies including mouse…” Suggested: “followed by incubation with the indicated primary antibodies including mouse…”

Response: Thank you so much for mentioning it. We have revised the descriptions accordingly in the Materials and methods section (Page 8, lines 131-132).

b. Page 13, line 120-121. “rabbit anti-Smad2 phosphorylation 121 (1:1000, Cell Signaling), ...” Suggested: “rabbit anti- phospho Smad2 (1:1000, Cell Signaling), …”

Response: Thank you for the great suggestion. We have revised the descriptions accordingly in the Materials and methods section (Page 8, line 133).

c. Page 14, line 138. “…and incubated with Smad2 (1:1000, Cell Signaling) antibodies at 4 °C.” For how long?

Response: Thank you for the excellent question. We have added the incubation time of primary antibodies in the Materials and methods section (Page 9, line 151).

d. The experimental design is not entirely clear. When does Leonurine begin to be applied to the animal?

Response: Thank you so much for mentioning it. We have enriched the details of animal experiments in the Materials and Methods section (Pages 5-6, lines 69-89).

e. Another issue concerns the in vitro study. There is no reference to cell culture and treatment. It is necessary to describe the culture conditions, medium, culture time. And the experimental treatment design.

Response: Thank you for your comment. We have added detailed information on in vitro study (Pages 7-8, lines 118-125).

3. Results

a. Page 16, line 164-169. “After ISO treatment, rats were administered leonurine for eight weeks. Levels of left ventricular end-diastolic pressure (LVEDP), its rising and falling rate (±dP/dt max), lactic dehydrogenase (LDH) and creatine kinase (CK) were significantly increased 1.53-, 2.01-, 2.31-, 1.48- and 1.41-fold, and left ventricular systolic pressure (LVSP) was decreased 1.71-fold in rats after ISO treatment (Fig 1 and 2).” I suggest rewriting this section. Lots of information about the measured parameters. It could put the numerical data in an ordering closer to parameter groups. Ex: left ventricular end-diastolic pressure (LVEDP), its rising and falling rate (±dP/dt max) increased as shown respectively 1.53-, 2.01- and 2.31.

Response: Thank you for your comment. We have revised the descriptions accordingly in the Results section (Pages 11-17, lines 178-322).

b. Page 17, line 183-184. “After leonurine treatment, expressions of proteins related to pyroptosis were decreased in the heart from rats induced by ISO (Fig 5).” Suggested: “After leonurine treatment, the expression of proteins related to…”

Response: Thank you for your comment. We have revised the descriptions accordingly in the Results section (Page 14, lines 245-246).

c. According to the figures, it is clear that there are different answers in relation to the concentrations of leonurine used. These differences are not addressed during the discussion and there is no information for the reader to choose the concentration used in the cell culture treatment. The descriptor text of the results does not even indicate when there is a change in the use of animal tissue for cell culture. Clarity in writing is necessary for the understanding of the article to be total.

Response: Thank you for your comment. According to your suggestion, we have supplemented the relevant information in the Discussion section (Page 18, lines 326-332) and Results section (Pages 14-17, lines 262-322).

4. Figures

a. Fig. 3 Areas of cardiac fibrosis in ISO-induced rats with leonurine treatment for eight weeks. Representative pictures of left ventricles from each group after H&E staining, Masson staining, and PSR staining (magnification, ×4, ×40). Leonurine decreased areas of collagen and fibers compared to the model group. (A) Collagen and fibers areas are pink after H&E staining. (B) Collagen and fibers areas are blue after Masson staining. (C) Collagen and fibers areas are yellow after PSR staining. The description of figure A is interchanged with that of figure B.

Response: Thank you for your comment. We have revised the figure legends of Fig 3 accordingly.

b. In figure 8 there is an error in the first line of image A. It should be cleaved Capase 1.

Response: Thank you for your comment. We have corrected these errors in Fig 9.

5. Considerations:

a. I suggest that a rereading of the article be done in order to make the writing clearer.

Response: We appreciate your suggestion. We are very sorry for the confused expressions in this article. We have reworked this article with the help of native English speakers.

b. The authors speak of the effect of leonurine in blocking the TGFbeta/SMAD-2 signaling pathway. An experiment in which leonurine was used and compared to a pathway inhibitor would be great.

Response: Thank you for your suggestion. Although the use of leonurine in the experiments and comparison with pathway inhibitors will better clarify the mechanism of leonurine in the treatment of pyroptosis, our preliminary study has obtained a novel conclusion that leonurine inhibited pyroptosis to attenuate cardiac fibrosis by regulating TGF-β/Smad2 signaling pathway by in vivo and in vitro study. According to your suggestion, we will further use the pathway inhibitors to conduct in-depth mechanism studies and added the related information as limitations in the Discussion section (Page 21, lines 392-394).

Reviewer #2: Leonurine inhibits cardiomyocyte pyroptosis against cardiac fibrosis via the TGF-β/Smad2 signaling pathway Reviewer

Recommendation and Comments

Major Concern:

Response: Thank you for your constructive advice. According to your suggestion, we have revised the title to “Leonurine inhibits cardiomyocyte pyroptosis to attenuate cardiac fibrosis via the TGF-β/Smad2 signaling pathway.”

2. Abstract: The citation (line 7): “The manuscript studied the novel mechanism…” please change for “This report…”.

Response: Thank you for your comment. We have revised the descriptions accordingly in abstract (Page 2, lines 7-8).

Response: Thank you for so much for your great reminder. We have added the related information in the Introduction section (Page 4, lines 50-57).

4. Materials and Methods:

a. Leuronine was obtained from Fudan University; to me it seems that the substance is not commercially available. Is it as extraction? The authors should provide more information.

Response: Thank you so much, it is a good suggestion for our study. We have added detailed information on leonurine source in the Materials and Methods section (Page 5, lines 61-63).

b. The authors should provide the total n number and mention if the treatment induced any loss of the animals.

Response: Thank you for the kind suggestion. According to your suggestion, we have enriched the details of animal experiments in the Materials and Methods section (Page 5, line 69 and Page 6, line 82).

Response: Thank you for your comment. According to your suggestion, we have revised the descriptions of treatment protocol in the Materials and Methods section (Pages 5-6, lines 69-89).

5. Results: The quality of Fig 3 is poor. The yellow mentioned in the text can´t be seen.

Response: We appreciate your suggestion. We have revised and updated a new Fig 3.

6. Discussion:

a. Paragraphs 1 and 2 says the same things, please be concise.

Response: Thank you so much for your kind reminder. According to your suggestion, we have revised the related information in the Discussion section (Page 18, lines 326-332).

Response: Thank you for your professional comment. ISO is a beta-adrenergic agonist. When β-adrenergic receptors are over-activated by ISO, membrane nanotubes promote inflammasome activation to spread from cardiomyocytes to cardiac fibroblasts, resulting in cardiac fibroblast pyroptosis [1]. β-receptor blockers such as atenolol exert their cardiovascular protective effects mainly by antagonizing β-adrenergic receptors, especially β1-receptor-mediated cardiotoxicity [2]. At present, there is no literature report on the treatment of pyroptosis by leonurine, but it has been confirmed that leonurine can inhibit the activation of NLRP3 inflammasome and reduce the release of inflammatory factors [3,4], which may be the potential mechanism of leonurine in the treatment of pyroptosis. Although there are currently no reports on the combined therapy of leonurine and β-adrenergic receptor blockers, we reviewed the literature and found that Higenamine, a benzylisoquinoline alkaloid, inhibited ISO-induced cardiac fibrosis independent of β2-adrenergic receptors [5]. Therefore, we speculate that leonurine, which also belongs to the alkaloid, may not exert its therapeutic effect through β-adrenergic receptors either. Therefore, we have revised the related information in the Discussion section (Page 19, lines 344-347).

Response: Thank you for your professional comment. In this study, ISO was used as an inducer to cause heart failure in rats, and then the effects and mechanism of leonurine on heart failure rats were explored. According to the chemical structure, both ISO and leonurine are rich in electron-donating solid groups such as amino groups, hydroxy, and phenolic hydroxyl, which makes the chemical combining difficult. And according to the pharmacokinetics, depending on the different administrations and metabolic rates of ISO (S.C., Tmax=14.00±5.48 min) [6] and leonurine (P.O., Tmax=51.6±15.6 min) [7], they are also difficult to ‘get adequate’ exposure to in vivo. Therefore, we speculated there might be no physical interaction between the two drugs. This study demonstrated that leonurine could improve hemodynamic parameters and the levels of LDH and CK in ISO-induced heart failure rats. According to reports, leonurine could also reduce hemodynamic parameters and the levels of LDH and CK in the myocardial infarction rats induced by coronary artery ligation [8] and the heart ischemia rats induced by ligation of the left coronary artery [9], thereby exerting cardioprotective effects similar to this study. Therefore, we speculate that the cardioprotective mechanism of leonurine may be independent of β-adrenergic receptors. We have revised the related information in the Discussion section (Page 18, lines 322-337).

d. At the end of the discussion, the authors mentioned “some limitations”, the authors should cite and discuss it better.

Response: Thank you for the great suggestion. We have added the limitations accordingly in the Discussion section (Page 18, lines 318-320).

Minor Concern:

1. Please, provide the p values of each result in order for a better conclusion of the reader.

Response: We appreciate your suggestion. We have supplemented the p value in the Results section (Pages 11-17, lines 178-322).

2. Please, revise Figure legends 1, 2, 3 and 4. The letters that label’s the panels are exchanged.

Response: Thank you so much for mentioning it. We have revised the descriptions accordingly in the figure legends of Fig 1, 2, 3, and 4.

3. It is encouraged that the authors draw a picture underling the proposed molecular mechanism of Leuronide.

Response: Thank you for your comment. According to your suggestion, we have supplemented a summary picture of the proposed molecular mechanism of leonurine as Fig 10.

Response: Thank you for your kind suggestion. We have revised the manuscript, refined the chapters in the Results section, and modified the language of the manuscript under the guidance of native English speakers.

References

[1] Shen J, Wu J-M, Hu G-M, Li M-Z, Cong W-W, Feng Y-N, et al. Membrane nanotubes facilitate the propagation of inflammatory injury in the heart upon overactivation of the β-adrenergic receptor. Cell Death Dis. 2020;11(11):1-11. doi: 10.1038/s41419-020-03157-7.

[2] Bencivenga L, Liccardo D, Napolitano C, Visaggi L, Rengo G, Leosco D. β-Adrenergic receptor signaling and heart failure: From bench to bedside. Heart Failure Clinics. 2019;15(3):409-19. doi: 10.1016/j.hfc.2019.02.009.

[3] Rong H, Yao C. Leonurine inhibits NLRP3 inflammasome hyperactivation and regulates macrophage M1/M2 phenotype differentiation. J Pharmaceut Pract. 2021;39(2):143-7. doi: 10.12206/j.issn.1006-0111.202101003.

[4] Zhang Q, Sun Q, Tong Y, Bi X, Chen L, Lu J, et al. Leonurine attenuates cisplatin nephrotoxicity by suppressing the NLRP3 inflammasome, mitochondrial dysfunction, and endoplasmic reticulum stress. Int Urol Nephrol. 2022:1-10. doi: 10.1007/s11255-021-03093-1.

[5] Zhu J-x, Ling W, Xue C, Zhou Z, Zhang Y-s, Yan C, et al. Higenamine attenuates cardiac fibroblast abstract and fibrosis via inhibition of TGF-Β1/Smad signaling. Eur J Pharmacol. 2021;900:174013. doi: 10.1016/j.ejphar.2021.174013.

[6] Zhou J, Yin H, Ma H, Wei S, Wen E, Zhang W, et al. Efficient and selective analytical method for the quantification of a β-adrenoceptor agonist, isoproterenol, by LC–MS/MS and its application to pharmacokinetics studies. J Liq Chromatogr R T. 2017;40(13):699-705. doi: 10.1080/10826076.2017.1348952.

[7] Li B, Wu J, Li X. Simultaneous determination and pharmacokinetic study of stachydrine and leonurine in rat plasma after oral administration of Herba Leonuri extract by LC–MS/MS. J Pharmaceut Biomed. 2013;76:192-9. doi: 10.1016/j.jpba.2012.12.029.

[8] Xu L, Jiang X, Wei F, Zhu H. Leonurine protects cardiac function following acute myocardial infarction through anti‑apoptosis by the PI3K/AKT/GSK3β signaling pathway. Mol Med Rep. 2018;18(2):1582-90. doi: 10.3892/mmr.2018.9084.

[9] Liu X, Pan L, Chen P, Zhu Y. Leonurine improves ischemia-induced myocardial injury through antioxidative activity. Phytomedicine. 2010;17(10):753-9. doi: 10.1016/j.phymed.2010.01.018.

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PLoS One. doi: 10.1371/journal.pone.0275258.r003

Decision Letter 1

Luis Eduardo M Quintas

13 Sep 2022

Leonurine inhibits cardiomyocyte pyroptosis to attenuate cardiac fibrosis via the TGF-β/Smad2 signalling pathway

PONE-D-22-05865R1

Dear Dr. Zhu,

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PLoS One. doi: 10.1371/journal.pone.0275258.r004

Acceptance letter

Luis Eduardo M Quintas

25 Oct 2022

PONE-D-22-05865R1

Leonurine inhibits cardiomyocyte pyroptosis to attenuate cardiac fibrosis via the TGF-β/Smad2 signalling pathway

Dear Dr. Zhu:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Hemodynamic variables.

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S2 Table. OD values of CK, LDH, and IL-1β.

(XLSX)

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S3 Table. Western blot analysis.

(XLSX)

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S4 Table. ROS fluorescence intensity.

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S1 Raw images. Original entire blots for figures.

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Leonurine inhibits cardiomyocyte pyroptosis to attenuate cardiac fibrosis via the TGF-β/Smad2 signalling pathway

Zhaoyi Li

Keyuan Chen

Yi Zhun Zhu

Roles

Abstract

Introduction

Materials and methods

Materials

Animal experiments

Haemodynamic measurement

Biochemical analysis

Histology analysis

Cell culture and treatments

Western blot analysis

Immunofluorescence staining

Measurement of ROS generation

Statistical analysis

Results

Leonurine improved cardiac fibrosis and exerted cardioprotective effects in vivo

Fig 1. Leonurine enhanced cardiac function in rats with ISO-induced heart failure by improving haemodynamic variables.

Fig 2. Leonurine reduced myocardial injury in rats with ISO-induced heart failure.

Fig 3. Leonurine reduced the area of cardiac fibrosis in rats with ISO-induced heart failure.

Fig 4. Leonurine reduced the expression of proteins related to fibrosis in heart tissues from rats with ISO-induced heart failure.

Leonurine inhibited pyroptosis in vivo

Fig 5. Leonurine reduced the expression of proteins related to pyroptosis in heart tissues from rats with ISO-induced heart failure.

Leonurine inhibited pyroptosis in vitro

Fig 6. Leonurine reduced the expression of proteins related to fibrosis in H9c2 cells stimulated by TGF-β.

Fig 7. Leonurine reduced the expression of proteins related to pyroptosis in H9c2 cells stimulated by TGF-β.

Leonurine inhibited cardiomyocyte pyroptosis via the TGF-β/Smad2 signalling pathway in vitro

Fig 8. Leonurine decreased ROS production by flow cytometric detection in H9c2 cells stimulated by TGF-β.

Fig 9. Leonurine inhibited pyroptosis by regulating TGF-β/Smad2 signalling pathway in H9c2 cells stimulated by TGF-β.

Discussion

Fig 10. Diagram of the potential mechanism of the effects of leonurine attenuating cardiac fibrosis in heart failure.

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Luis Eduardo M Quintas

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Author response to Decision Letter 0

Decision Letter 1

Luis Eduardo M Quintas

Roles

Acceptance letter

Luis Eduardo M Quintas

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Associated Data

Supplementary Materials

Data Availability Statement

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