Salvianolic acid B alleviates myocardial ischemic injury by promoting mitophagy and inhibiting activation of the NLRP3 inflammasome
- Authors:
- Published online on: October 14, 2020 https://doi.org/10.3892/mmr.2020.11589
- Pages: 5199-5208
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Copyright: © Hu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Cardiovascular disease is a major health issue worldwide, with acute myocardial ischemia accounting for >40% deaths in China (1). Severe myocardial ischemia may result in a characteristic pattern of metabolic and ultrastructural changes (2), which can lead to irreversible injury. Although several studies have been performed, the molecular mechanisms underlying myocardial ischemic injury remain unclear (3,4). In addition, it has been reported that the occurrence of myocardial ischemia is often accompanied by severe inflammatory response (5) and activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome (6).
The NLRP3 inflammasome is a cytosolic protein complex comprising NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1. Notably, the NLRP3 inflammasome is assembled in response to several endogenous “danger signals”, such as oxidative stress, lysosome destabilization and mitochondrial dysfunction (7). Recently, it has been reported that activation of the NLRP3 inflammasome is associated with the pathogenesis of numerous inflammatory diseases (8,9), and that it may regulate the secretion of several pro-inflammatory cytokines, such as IL-1β and IL-18 (10). Furthermore, inhibition of the NLRP3 signaling pathway has been shown to protect against myocardial ischemic injury induced by coronary artery ligation (11).
Sirtuin 1 (SIRT1) is a member of the sirtuin class III histone deacetylase family. It serves important roles in several important biological processes, including mitophagy, cell senescence and cell apoptosis (12). In recent years, several studies have focused on the role of SIRT1-mediated mitophagy in the regulation of NLRP3 activation. These studies suggested that SIRT1-mediated mitophagy is a negative regulator of NLRP3 inflammasome activation (13) and could significantly protect against myocardial ischemic injury (14). However, whether salvianolic acid B (Sal B) could alleviate myocardial ischemic injury by promoting SIRT1-mediated mitophagy remains unclear.
Salvia miltiorrhiza Bge has been used to treat myocardial ischemia in mainland China for >2,000 years. Compared with chemical drugs, this plant has numerous advantages, including multi-ingredients (which enables less opportunity for resistance), multi-targets and few side effects (15,16). As the main active water-soluble substance in S. miltiorrhiza Bge, Sal B has been reported to exert several pharmacological activities, including antitumor (17), anti-inflammatory (18) and antioxidant (19) effects. It has previously been reported that Sal B may exert protective effects against myocardial ischemic injury (20). However, whether Sal B could regulate mitophagy and inhibit activation of the NLRP3 inflammasome during myocardial ischemic injury remains unclear. Therefore, the present study investigated whether Sal B could alleviate myocardial ischemic injury by promoting cell mitophagy and inactivating the NLRP3 inflammasome.
Materials and methods
Materials
Sal B was from Shanghai Yuanye Biotechnology (cat. no. B20261). Isoproterenol (ISO) was obtained from Beijing Solarbio Science & Technology Co., Ltd. (cat. no. II0200). Dulbecco's modified Eagle's medium (DMEM) containing high glucose was obtained from Wisent Biotechnology. Trypsin was obtained from Beyotime Institute of Biotechnology. The creatine kinase isoenzyme MB (CK-MB; cat. no. H197) and glutamic oxaloacetic transaminase (GOT; cat. no. C010-2-1) ELISA kits were obtained from Nanjing Jiancheng Bioengineering Institute. Evagreen 2X quantitative PCR (qPCR) MasterMix-Low Rox kit and the IL-1β ELISA kit (cat. no. K4796-100) were purchased from Abmgoodchina, Inc. and BioVision, Inc., respectively. Adenosine triphosphate (ATP) was obtained from Amresco, LLC. Lipopolysaccharide (LPS) was obtained from Cell Signaling Technology, Inc. Primary antibodies against SIRT1 (cat. no. ab189494), microtubule-associated protein 1A/1B-light chain 3 (LC3; cat. no. ab48394), P62 (cat. no. ab91526), PTEN-induced kinase 1 (PINK1; cat. no. ab23707), Beclin-1 (cat. no. ab62557), NLRP3 (cat. no. ab214185), IL-1β (cat. no. ab205924) were purchased from Abcam, and primary antibodies against Parkin (cat. no. 14060-1-AP), caspase-1 (cat. no. 22915-1-AP) and GAPDH (cat. no. 10494-1-AP) were purchased from Proteintech Group, Inc. The distilled water used in this study was purified using a Milli-Q system (EMD Millipore). All culture plates were purchased from Corning, Inc.
Animals
Male Sprague-Dawley (SD) rats (age, 2 months; weight, 180–220 g), were housed in pathogen-free conditions under a 12-h light/dark cycle at 22–24°C (humidity, 40–70%) with ad libitum access to a normal pellet diet and clean water. The protocol of the present study was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (Nanjing, China; approval no. 201906A036).
A total of 50 male SD rats were randomly divided into the following five groups (n=10/group): i) control group; ii) model group; iii) model + Sal B (5 mg/kg) group; iv) model + Sal B (10 mg/kg) group; and v) model + Sal B (15 mg/kg) group. The rats in the Sal B treatment groups received a daily intraperitoneal injection of Sal B (dissolved in PBS) for 7 consecutive days, whereas rats in the control and model groups were treated with equal amounts of normal saline. Furthermore, the acute myocardial ischemia model was induced by hypodermic injection of ISO (30 mg/kg/d) once daily from the 5th day. Notably, the modelling method of ISO-induced myocardial ischemia has previously been confirmed to be effective (21,22). Following treatment with Sal B for 24 h on the 7th day, all rats were anesthetized by intraperitoneal injection of pentobarbital sodium solution (50 mg/kg) and a total of 2 ml blood was withdrawn from the abdominal aorta. Subsequently, animals were euthanized with 5% pentobarbital sodium solution (150 mg/kg), and the hearts were harvested for a series of pathological and biochemical studies. Euthanasia should result in rapid loss of consciousness, followed by respiratory and cardiac arrest, and ultimate loss of all brain function. Death was confirmed by checking the breathing rate, heartbeat and pupillary reflexes after euthanasia and prior to disposal of the animal.
Determination of ST-segment elevation
Following anesthesia by intraperitoneal injection of 50 mg/kg sodium pentobarbital, the electrocardiogram (ECG) indexes of rats were determined using a power-lab system (ADInstruments, Ltd.). Myocardial ischemia was verified by ST-segment elevation recorded by ECG and the results were expressed as relative to the control.
Hematoxylin and eosin (H&E) staining
The heart tissues were fixed in 10% neutral buffered formalin for 24 h at room temperature, embedded in paraffin and were then sliced into 5-µm conventional sections using a microtome. Subsequently, the embedded blocks were prepared into pathological sections. The sections were stained with H&E for 5 min at room temperature. The sections were then mounted and histologically observed under a light microscope (magnification, ×200).
Determination of serum CK-MB, GOT and IL-1β concentrations
Blood samples were collected from the abdominal aorta of each rat 24 h after Sal B administration on the 7th day and the serum was obtained by centrifugation at 1,500 × g for 10 min at room temperature. Subsequently, the serum levels of CK-MB, GOT and IL-1β were determined using a microplate system (BioTek Instruments, Inc.) and the aforementioned ELISA kits, according to the manufacturer's instructions.
Reverse transcription (RT)-qPCR
Total RNA was extracted from heart tissues using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and RNA concentration was determined using a NanoDrop (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using an 5X All-in-one RT MasterMix kit (Abmgoodchina Inc.), according to the manufacturer's instructions. Subsequently, qPCR was performed using the Evagreen 2X qPCR MasterMix-Low Rox kit (Abmgoodchina Inc.) on the ABI 7500 Fast Real-Time PCR Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 10 min; followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The mRNA expression levels of NLRP3, ASC, caspase-1 and IL-1β were determined using the 2−ΔΔCq method (23) and were normalized to GAPDH expression levels. The sequences of the specific primers used for qPCR analysis are listed in Table I.
Cell culture and treatment
The H9C2 cell line, which is widely used to study myocardial ischemia (24,25), was obtained from the American Type Culture Collection. Cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO2 atmosphere.
H9C2 cells were seeded into a 6-well plate at a density of 5×104 cells and cultured overnight. Subsequently, the cells were randomly divided into the following five groups: i) Control group; ii) LPS + ATP group; iii) LPS + ATP + Sal B (1 µM) group; iv) LPS + ATP + Sal B (5 µM) group; and v) LPS + ATP + Sal B (25 µM) group. Cells in Sal B treatment groups were pretreated with different concentrations of Sal B (1, 5 and 25 µM) for 24 h at 37°C. After discarding the supernatant, all cells, except within the control group, were washed twice with PBS and stimulated with LPS (1 µg/ml) for 24 h at 37°C. Subsequently, cells were stimulated with ATP (5 mM) for 2 h at 37°C. Cells in the control group were cultured normally (high-glucose DMEM supplemented with 10% FBS at 37°C). Finally, all cells in all groups were collected for subsequent analyses.
Western blot analysis
Cultured H9C2 cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) and the total protein concentration was quantified using a BCA assay (Beyotime Institute of Biotechnology). Total protein samples (50 µg) were separated by SDS-PAGE on 6–10% gels. The total protein samples were then electrotransferred onto polyvinylidene fluoride membranes. After blocking with 5% non-fat milk in TBS-0.05% Tween-20 for 1 h at room temperature, the membranes were incubated with primary antibodies against NLRP3 (1:1,000), caspase-1 (1:1,000), IL-1β (1:1,000), SIRT1 (1:1,000), P62 (1:1,000), Parkin (1:1,000), PINK1 (1:1,000), Beclin-1 (1:1,000), LC3 (1:1,000) and GAPDH (1:5,000) at 4°C overnight with gentle agitation. Subsequently, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (dilution, 1:1,000; Beyotime Institute of Biotechnology; cat. no. A0208) in non-fat milk for 1 h at room temperature. The membranes were visualized using an ECL kit (Beyotime Institute of Biotechnology) and densitometric analysis was performed using Image Lab software version 4.0.1 (Bio-Rad Laboratories, Inc.)
Measurement of intracellular reactive oxygen species (ROS) generation
The intracellular ROS levels were measured using 2,7-dichlorofluorescin diacetate (DCFH-DA; cat. no. S0033S; Beyotime Institute of Biotechnology). In the presence of ROS, DCFH-DA is oxidized into fluorescent dichlorofluorescein (DCF) (26). After treatment, the collected cells (1×106/well) were resuspended in 1 ml DCFH-DA reagent (10 µM) and incubated at 37°C for 30 min in the dark. Images were captured using a fluorescence microscope (magnification, ×200) and the mean density was analyzed using ImageJ software version 1.8.0 (National Institutes of Health) using the following formula: Mean density = integrated density/total area. The fluorescence intensity of DCF was measured by flow cytometry (BD LSR II; BD Biosciences) and analyzed using FACSDiva software version 4.1 (BD Biosciences).
TUNEL assay
Apoptosis was analyzed using the One Step TUNEL Apoptosis Assay kit (cat. no. C1090; Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Briefly, H9C2 cells were seeded into a 6-well plate at a density of 5×104 cells. Cells were co-treated with Sal B and LPS + ATP (stimulation) as aforementioned. Subsequently, cells were fixed in 4% paraformaldehyde for 30 min at 37°C, permeabilized in 0.1% Triton X-100 for 2 min and incubated with TUNEL assay reagents for 1 h at 37°C. Images were captured using a fluorescence microscope and fluorescence was measured using a fluorescence spectrophotometer (absorbance wavelength, 550 nm). The mean density was determined as previously described above.
Mitochondrial membrane potential (MMP) measurement
An MMP assay kit with JC-1 (cat. no. C2006; Beyotime Institute of Biotechnology) was used to evaluate MMP, according to the manufacturer's protocol. Briefly, 1×106 cells/well were incubated with JC-1 for 30 min at 37°C, rinsed twice with PBS and the staining images were captured under a fluorescence microscope. The MMP was indicated by the ratio of red/green fluorescence intensity.
Statistical analysis
Experiments were repeated ≥3 times. Data are presented as the mean ± standard deviation. The statistically significant differences between the means of multiple groups were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. All statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.
Results
Echocardiography of rats with acute myocardial ischemia
Standard echocardiography was performed at room temperature for all groups of rats 24 h after the administration of Sal B on the 7th day. Control rats exhibited a normal ECG pattern with regular sinus rhythm (Fig. 1). However, an irregular sinus rhythm with significant elevation of the ST-segment was observed in the ECGs of ISO-induced model rats compared with those of control rats. Furthermore, 10 and 15 mg/kg Sal B treatment markedly reversed these ISO-induced alterations (Fig. 1).
Pathological observation
The results of H&E staining revealed that the hearts of rats in the control group maintained a normal form and function (Fig. 2). However, the hearts of rats in the model group exhibited obvious rupture and lysis of myocardial fibers, accompanied by marked inflammatory cell infiltration. Notably, treatment with Sal B alleviated these pathological observations (Fig. 2).
Sal B reduces the levels of CK-MB, GOT and IL-1β in the serum of rats with myocardial ischemia
Elevation in the serum levels of cardiac markers, such as CK-MB and GOT, is an important basis for the diagnosis of acute myocardial ischemia (27). To evaluate the effects of Sal B on myocardial ischemic injury, the serum levels of CK-MB, GOT and IL-1β were determined. As shown in Fig. 3, intraperitoneal injection of ISO significantly increased the levels of CK-MB, GOT and IL-1β compared with those in the control group; treatment with Sal B (5, 10 and 15 mg/kg) significantly reversed the levels of these factors.
Effects of Sal B on the mRNA expression levels of NLRP3, ASC, caspase-1 and IL-1β in rats with ISO-induced myocardial ischemia
To investigate whether Sal B could inhibit the expression of the NLRP3 inflammasome in rats with ISO-induced myocardial ischemia, RT-qPCR analysis was conducted to determine the mRNA expression levels of the related genes. ISO stimulation significantly increased the mRNA expression levels of the NLRP3 inflammasome components compared with those in the control group (Fig. 4). However, the expression levels of NLRP3, ASC, caspase-1 and IL-1β in the Sal B-treated group (15 mg/kg) were significantly reduced compared with those in the model group (Fig. 4).
Sal B treatment promotes SIRT1-mediated mitophagy
The contraction and functioning of the heart are mainly dependent on mitochondria to provide energy. A previous study demonstrated that myocardial ischemic injury is often accompanied by mitochondrial dysfunction (28). Mitophagy is considered an effective process to remove damaged mitochondria and maintain normal mitochondrial function (29). To investigate the effect of Sal B on mitophagy during myocardial ischemic injury in cardiomyocytes, the expression levels of the proteins SIRT1, PINK1 and Parkin and were detected in H9C2 cells (Fig. 5). The differences in the protein expression levels of SIRT1 and Parkin in the control and LPS + ATP groups were not statistically significant; however, the expression levels of SIRT1 and Parkin were significantly decreased in the LPS + ATP group compared with those in the Sal B-treated groups (5 and 25 µM). Furthermore, LPS + ATP stimulation significantly increased the expression levels of PINK1 compared with the control group; however, Sal B treatment (25 µM) significantly reduced the expression levels. In addition, the expression levels of autophagy-related proteins P62, LC3 and Beclin-1 were evaluated. Notably, LPS + ATP stimulation significantly increased the expression level of autophagy substrate P62 compared with the control group; however, Sal B treatment (25 µM) significantly reduced the expression levels. Furthermore, LPS + ATP significantly upregulated the expression levels of cellular autophagy markers Beclin-1 and LC3-II compared with those in the control group. The expression levels of Beclin-1 following Sal B treatment (25 µM) and LC3-II following Sal B treatment (5 and 25 µM) were significantly increased compared with those in LPS + ATP group, thus suggesting that Sal B (25 µM) may mediate the induction of mitophagy and autophagy in myocardial ischemic injury.
Sal B inhibits LPS + ATP-induced ROS production
Excessive ROS production has been associated with impaired mitochondrial function and increased H9C2 cell apoptotic rate (30). As shown in the fluorescence images in Fig. 6, LPS + ATP stimulation significantly increased the fluorescence intensity compared with the control group, indicating the production of ROS. Conversely, pretreatment with Sal B attenuated the DCF fluorescence induced by LPS + ATP. Consistent with the fluorescence results, flow cytometric analysis demonstrated that the levels of intracellular ROS production in the LPS + ATP group were notably increased compared with those in the control group, whereas pretreatment with Sal B reversed this effect (Fig. 6).
Effects of Sal B on MMP
A previous study reported that decreased MMP is closely associated with mitochondrial dysfunction (31). To investigate whether Sal B could inhibit mitochondrial dysfunction by increasing MMP, a JC-1 kit was used. The JC-1 red/green fluorescence ratio was used as a parameter to estimate changes in MMP. The results revealed that the MMP in H9C2 cells was significantly reduced by LPS + ATP stimulation compared with in the control group, whereas Sal B pre-treatment (5 and 25 µM) significantly increased its potential (Fig. 7).
Effects of Sal B on activation of the NLRP3 inflammasome
A previous study suggested that the occurrence of mitophagy may lead to degradation of the NLRP3 inflammasome, which in turns prevents the activation of NLRP3 inflammasome signaling and reduces the secretion of mature IL-1β (32). To explore whether Sal B treatment could attenuate the expression levels of the NLRP3 inflammasome components, the protein expression levels of NLRP3, caspase-1 and IL-1β were evaluated. The results demonstrated that the protein expression levels of NLRP3, caspase-1 and IL-1β were significantly increased in H9C2 cells following LPS + ATP stimulation; however, pre-treatment with Sal B (25 µM) significantly reduced the expression levels of these proteins (Fig. 8).
Effects of Sal B on the apoptosis of H9C2 cells
Assembly of the NLRP3 inflammasome may activate caspase-1 and promote apoptosis (33). As shown in Fig. 9, LPS + ATP stimulation significantly promoted apoptosis compared with in the control group, whereas Sal B pre-treatment (5 µM) significantly reversed this effect.
Discussion
In the present study, Sal B markedly attenuated ISO-induced acute myocardial ischemic injury in rats and reduced the serum levels of several cytokines, including CK-MB, GOT and IL-1β. Furthermore, to the best of our knowledge, the present study was the first to demonstrate that Sal B treatment may be able to maintain mitochondrial function, promote SIRT1-mediated mitophagy and inhibit activation of the NLRP3 inflammasome. However, the present study did not provide much information on cardiac pathology and function; we aim to assess these further in future experiments.
It is widely accepted that myocardial ischemia is often accompanied by severe inflammatory response (34,35). As the core target of inflammatory responses, the NLRP3 inflammasome has been considered the key regulator of numerous inflammatory diseases (36). LPS + ATP is a classical activator of the NLRP3 inflammasome; it is widely used in studies of NLRP3 inflammasome-related diseases, including myocardial ischemia (37,38). Activation of the classic NLRP3 inflammasome requires two steps: Firstly, microorganisms or other inflammatory factors bind to Toll-like receptor 4 (TLR4) and activate the TLR4/NF-κB signaling pathway to induce the expression of NLRP3, pro-caspase-1 and pro-IL-1β (priming phase). Secondly, extracellular ATP or other stimuli, such as lysosome destabilization and mitochondrial dysfunction (39), directly stimulate the activation of caspase-1, thereby leading to the release of IL-1β and IL-18 (triggering phase). Activation of the NLRP3 inflammasome in cardiomyocytes requires the involvement of both priming and triggering phases (40). A previous study in our laboratory reported that Sal B exerted its cardioprotective effects by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway (41), and myeloid differential protein-2 was considered the potential target. However, owing to the multi-target characteristics of natural products, it was hypothesized that Sal B may inhibit the NLRP3 inflammasome through other mechanisms. Therefore, the present study explored whether Sal B could inhibit the triggering phase of the NLRP3 inflammasome, thereby attenuating myocardial ischemic injury.
The contraction and functioning of the heart are mainly dependent on mitochondria to provide energy. Emerging evidence has suggested that the accumulation of dysfunctional mitochondria may result in increased intracellular ROS, thus leading to cardiac dysfunction (42). Mitophagy is an effective process that can remove damaged mitochondria and maintain mitochondrial function (43). The association between SIRT1 and mitophagy has also been previously reported. For example, it has been reported that the SIRT1-PINK1-Parkin axis may participate in resveratrol-induced mitophagy and cardioprotection (44). Furthermore, it has been revealed that promoting mitophagy may negatively regulate activation of the NLRP3 inflammasome, which may exert significant protective effects against myocardial ischemia (45,46). In conclusion, the present study demonstrated that Sal B could significantly inhibit intracellular ROS production, increase the mitochondrial membrane potential and regulate the protein expression levels of SIRT1, Parkin and PINK1 in myocardial ischemia, which in turn could mediate the promotion of mitophagy and inactivation of the NLRP3 inflammasome. Therefore, Sal B, as a natural product with little toxicity, may provide a novel therapeutic strategy for the treatment of myocardial ischemia.
Acknowledgements
Not applicable.
Funding
The present study was supported by The Jiangsu Administration of Traditional Chinese Medicine (grant no. ZD301701).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
YH and XW contributed equally to this work. LX designed this project. YH, XW, YP and QL performed the experiments and analyzed the data. YH and XW contributed to the writing of the manuscript. All authors contributed to the revision of this manuscript, and read and approved the final manuscript.
Ethics approval and consent to participate
The protocol was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (approval no. 201906A036).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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