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The Kaohsiung Journal of Medical Sciences logoLink to The Kaohsiung Journal of Medical Sciences
. 2022 Nov 21;39(3):290–301. doi: 10.1002/kjm2.12627

Total saponins of Aralia elata (Miq.) Seem. alleviate myocardial ischemia‐reperfusion injury by promoting NLRP3‐inflammasome inactivation via PI3K/Akt signaling

Li Sun 1,✉, Wei‐Xing Lu 2, Hui Li 1, Ding‐Ya Feng 1, Jing‐Xiao Nie 1
PMCID: PMC11895957  PMID: 36408810

Abstract

Total saponins of Aralia elata (Miq.) Seem. (TSAE) have been shown to play a significant role in cardiovascular protection, anti‐tumor, liver protection, anti‐oxidant stress, and anti‐inflammation. However, the specific mechanisms of TSAE in myocardial ischemia‐reperfusion injury (MIRI) remain largely elusive. Hearts from male Wistar rats were used to establish the isolated heart MIRI model. Using a multichannel physiological recorder, the whole course heart rate (HR), left ventricular development pressure (LVDP), and maximum rise/decrease rate of left ventricular pressure (±dp/dt max) were recorded. 2,3,5‐triphenyl‐2H‐tetrazolium chloride staining observed the infarct area, while hematoxylin & eosin staining detected pathological changes in myocardial tissue. Creatine kinase, lactate dehydrogenase, total superoxide dismutase, and malondialdehyde concentrations were determined by enzyme‐linked immunosorbent assay. Immunohistochemistry, quantitative PCR, and western blot assay were used to assess the amounts of IL‐18 and IL‐1β, NLR family protein (NLRP3) inflammasome‐ and apoptosis‐related proteins, respectively. Treatment with TSAE or MCC950 (NLRP3‐specific inhibitor) significantly reduced the myocardial infarction area, alleviated pathological changes in myocardial tissues, enhanced LVDP and ±dp/dt max levels, prevented myocardial oxidative damage, and inhibited NLRP3 inflammasome formation. In addition, TSAE enhanced Akt and GSK3β phosphorylation, and LY29004 co‐reperfusion markedly diminished the protective role of TSAE reperfusion on cardiac function, oxidative damage, and inflammatory responses. Collectively, TSAE treatment exhibited a protective effect on I/R‐triggered inflammatory responses, cell necrosis, and oxidative stress injury by stimulating PI3K/Akt signaling‐mediated NLRP3 inflammasome inhibition.

Keywords: myocardial ischemia‐reperfusion injury, NLRP3 inflammasome, PI3K/Akt signaling, total saponins of Aralia elata (Miq.) Seem.

Abbreviations

AMI

acute myocardial infarction

AS

Aralia elata (Miq.) Seem

CABG

coronary artery bypass grafting

CK

creatine kinase

±dp/dt max

maximum rise/decrease rate of left ventricular pressure

ELISA

enzyme‐linked immunosorbent assay

H&E

hematoxylin & eosin

HR

heart rate

IL

interleukin

ITT

intravenous thrombolytic therapy

LDH

lactate dehydrogenase

LVDP

left ventricular development pressure

MDA

malondialdehyde

MIRI

myocardial ischemia‐reperfusion injury

NLRP3

NLR family protein

PCI

percutaneous coronary intervention

SOD

total superoxide dismutase

TSAE

total saponins of Aralia elata (Miq.) Seem

1. INTRODUCTION

Acute myocardial infarction (AMI) is a life‐threatening acute cardiovascular disease in which prolonged ischemia and hypoxia due to acute coronary artery occlusion impair cardiac function. 1 Timely, rapid, and effective reperfusion therapies, such as intravenous thrombolytic therapy, percutaneous coronary intervention, and emergency coronary artery bypass grafting, at the onset of AMI are the keys to saving patients' lives. 2 , 3 However, sudden restoration of blood flow is usually accompanied by additional myocardial damage and cardiac electrical dysfunction, clinically referred to as myocardial ischemia‐reperfusion injury (MIRI). 4 Studies have shown that MIRI significantly affects the outcome of AMI patients and is one of the risk factors for high mortality in AMI patients. 5 However, there is still no effective way to avoid MIRI. Therefore, it is crucial to understand the underlying molecular mechanisms of MIRI and develop novel therapeutic strategies for its prevention and treatment.

Inflammasomes are large multiprotein complexes that initiate an organism's immune response by recognizing endogenous and exogenous disease risk signals. 6 , 7 The most extensively studied protein in the NLRs family is NOD‐like receptor protein 3 (NLRP3), which, as studies reveal, triggers inflammasome assembly by interacting with ASC and recruiting pro‐Caspase‐1; thus, promoting a strong immune response, including Caspase‐1 generation and the maturation and secretion of pro‐inflammatory cytokines such as interleukin (IL)‐18 and IL‐1β. 6 , 8 Currently, numerous studies have reported the vital role of the NLRP3‐inflammasome in inflammation‐related diseases, such as cancer, diabetes, cardiovascular disease, and so forth. 9 , 10 , 11 Targeting the increased NLRP3‐inflammasome during MIRI has been shown to effectively reduce the myocardial inflammatory response and infarct size. 12 , 13 Moreover, MCC950, a small‐molecule inhibitor targeting the NLRP3 inflammasome, significantly reduced fibrosis by inhibiting early inflammatory responses and improving cardiac function. 14 These findings indicate that the NLRP3‐inflammasome is widely considered a therapeutic target for MIRI treatment.

Aralia elata (Miq.) Seem. (AS) is a common medicinal plant distributed in South America and northeast China. 15 Its roots are widely used in the treatment of stomach ulcers, diabetes, arthritis, myocardial infarction, and other diseases. Several saponins have so far been isolated from the root, stem, leaves, and other parts of AS, which exhibit strong anti‐tumor, anti‐inflammatory, and anti‐myocardial ischemia functions. 16 , 17 It has recently been reported that the total saponins of Aralia elata (Miq.) Seem. (TSAE), the main pharmacological components of AS, have a protective effect on MIRI. According to Wang et al., TSAE administration significantly reduced the size of myocardial infarct and delayed myocardial pathological progression, which were linked to the suppression of calcium homeostasis imbalance and endoplasmic reticulum stress. 15 However, the specific molecular mechanism of TSAE in MIRI still remains unclear.

Given the preceding context, the goal of the current study was to determine whether TSAE targeting MIRI was associated with the NLRP3 inflammasome. A previous study revealed that TNF‐α‐mediated endothelial cell damage involved PI3K/Akt and NF‐κB signaling, which were the downstream pathways of TSAE. 18 Notably, the activation of PI3K/Akt signaling could inhibit the formation of NLRP3 inflammasome. 19 , 20 Similar findings reported in MIRI demonstrated that piperine‐mediated phosphorylation of Akt could suppress NLRP3‐mediated pyroptosis and protect the heart from I/R‐induced inflammatory injury, 21 thus suggesting a potential association of TSAE, PI3K/Akt, and NLRP3 inflammasomes in MIRI. To validate the hypothesis, a rat model of MIRI was established, and a series of experiments involving myocardial pathological staining, hemodynamics, activation of NLRP3‐inflammasome, and PI3K/Akt pathway were conducted. Finally, our data revealed that TSAE inhibited NLRP3 inflammasome activation in a PI3K/Akt signaling‐dependent manner, thus ameliorating the progression of MIRI.

2. METHODS

2.1. Animal preparation

Male Wistar rats (SPF, 250–280 g) acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were housed at 25 ± 2°C, with a 12‐h light /12‐h dark cycle and fed ad libitum with standard chow and water. All experimental procedures used in the current study have been approved by the Animal Experiments Ethics Committee of Dongfang Hospital, Beijing University of Chinese Medicine (approval number: 18–16).

2.2. Drug preparation

To prepare K‐H solution, the following ingredients were weighed and dissolved in deionized water until a total volume of 1 L was obtained: NaCl 6.903 g, NaHCO3 2.190 g, KCl 0.350 g, KH2PO3 0.163 g, MgSO4 0.144 g, anhydrous glucose 1.998 g, and CaCl2 0.139 g (pH: 7.35–7.45). TSAE was obtained from the Jilin Institute of Traditional Chinese Medicine (Jilin, China). MCC950 (a NLRP3 inflammasome‐specific inhibitor) and LY29004 (a PI3K/Akt signaling‐specific inhibitor) were acquired from MedChemExpress (New Jersey; 26846, 28157). These drugs were all dissolved in K‐H solution: (1) 5 mg TSAE was added to 1 L of K‐H solution to prepare 5 mg/L TASE solution; (2) 4.610 mg of LY29004 was dissolved in 0.3 ml of DMSO to obtain 15 μmol/L LY29004 using K‐H solution; and (3) 6.067 mg MCC950 was dissolved in 1 L of K‐H solution to acquire a concentration of 15 μmol/L.

2.3. Establishment of an I/R rat model

All rats underwent 3 days of conventional experimental feeding before being utilized to establish the Langendorff model. Briefly, 1000 U/kg of heparin saline was administered intraperitoneally to the rats after weighting. A 3% pentobarbital sodium (30 mg/kg, intraperitoneal injection) was used to anesthetize the rats 30 min after injection while they were fixed on a rat board. The left thorax was opened, the heart was dissected intact, and immediately placed in pre‐chilled K‐H solution. The perfusion tube was inserted above the aortic valve and coronary artery openings and secured with cotton thread, and K‐H solution was retrogradely injected. Next, a 5–0 silk suture was used to ligate the left anterior descending (LAD) coronary artery. Once the heart had resumed beating, a self‐made intraventricular pressure balloon tube was inserted into the left ventricle and connected to an MP150 multichannel physiologic recorder (Biopac). After 20 min of balancing, a normal index was recorded as the baseline. A heart rate (HR) of at least 220 beats/min and a left ventricular systolic pressure of at least 60 mmHg were the inclusion criteria of the model.

2.4. Experimental groups

In this study, rats were divided into the following groups (n = 12 per group) at random: (1) Sham group: Continuous balanced irrigation with K‐H solution for 125 min without ligation; (2) I/R group: balance for 20 min + LAD coronary artery ligation for 30 min + K‐H solution reperfusion for 75 min; (3) I/R + TSAE group: balance for 20 min + LAD coronary artery ligation for 30 min + 5 mg/L TSAE reperfusion for 75 min; (4) I/R + MCC950 group: balance for 20 min + LAD coronary artery ligation for 30 min + 15 μmol/L MCC950 reperfusion for 75 min; (5) I/R + LY29004 group: balance for 20 min + LAD coronary artery ligation for 30 min + 15 μmol/L LY29004 reperfusion for 75 min; and (6) I/R + TSAE + LY29004 group: balance for 20 min + LAD coronary artery ligation for 30 min + 15 μmol/L LY29004 reperfusion for 15 min + 5 mg/L TSAE reperfusion for 60 min. The specific groupings are shown in Figures 1A and 3A.

FIGURE 1.

FIGURE 1

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TSAE inhibited NLRP3 inflammasome activation in the MIRI rat model. (A) Schematic representation of animal grouping and handling. (B) Hemodynamic changes during cardiac reperfusion in rats. (C) Myocardial infarction size in rats as determined by TCC staining. (D‐E) Measurement of IL‐18 and IL‐1β expression levels in each group by quantitative PCR (qPCR) and immunohistochemical staining. (F‐G) qPCR and western blot assays for quantifying the changes in expression of NLRP3 inflammasome‐associated markers, including NLRP3, ASC, and Caspase‐1. Experimental data were shown as mean ± standard deviation, n = 12. LVEDd, left ventricular end‐diastolic diameter; LVEF, left ventricular ejection fractions; LVFS, left ventricular fractional shortening; LVESd, left ventricular end‐systolic diameter; TSAE, total saponins of Aralia elata (Miq.) Seem. *p < 0.05; **p < 0.01; and ***p < 0.001.

FIGURE 3.

FIGURE 3

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TSAE promoted the Akt and GSK‐3β phosphorylation in the myocardial ischemia‐reperfusion injury model. (A) Schematic representation of animal handling and grouping. (B‐C) Akt and GSK3β phosphorylation measured using a western blot. Data were expressed as mean ± standard deviation. N = 12 per group. TSAE, total saponins of Aralia elata (Miq.) Seem. *p < 0.05.

2.5. Hemodynamic detection

The HR, left ventricular end‐diastolic terminal pressure, and left ventricular end‐systolic pressure in each group were recorded by the MP150 multichannel physiologic recorder (Biopac), followed by the calculation of left ventricular developed pressure (LVDP), maximal left ventricular pressure rising rate (+dp/dt max) and maximal left ventricular pressure descending rate (−dp/dt max).

2.6. 2,3,5‐triphenyl‐2H‐tetrazolium chloride staining

After reperfusion, the heart was removed, stored in a refrigerator at −20°C for 20 min, and then cut into six pieces (approximately 2 mm thick) from the apex to the base of the heart. The slices were then stained with 1% 2,3,5‐triphenyl‐2H‐tetrazolium chloride (TTC) solution (NOVON, SS1747) at 38°C for 30 min and fixed overnight in 4% paraformaldehyde, after which they were photographed with a digital camera. Normal myocardium is red after staining, while infarcted myocardium is grayish‐white.

2.7. Hematoxylin and eosin staining

After reperfusion, the hearts were collected and fixed overnight in 4% paraformaldehyde. The fixed heart tissue was then washed, dehydrated, embedded in paraffin, and cut into 5 μm paraffin slices. Next, the slices were dewaxed and hydrated with decreasing concentrations of ethanol. The nuclei were stained with hematoxylin for 5 min and the cytoplasm with eosin for 3 min. Following that, the slices were dehydrated with a gradient of ethanol, cleared with xylene, and sealed with a neutral resin. Finally, the images were observed under a light microscope.

2.8. Immunohistochemical staining

After baking at 65°C for 20 min, the paraffin slices were dewaxed using xylene and alcohol solution with gradient reduction, followed by the addition of 0.01 mol/L (pH 6.0) citrate buffer for antigen repair. The slices were treated with 3% hydrogen peroxide solution to block endogenous peroxidase, followed by blocking with 5% BSA solution for 30 min after 15 min had passed. After that, the slices were incubated overnight at 4°C with primary antibodies including activated‐Caspase‐3 (1:150, PAA626Ra01, Cloud‐Clone CorP., Wuhan, China), Bax (1:50, PAB343Ra01, Cloud‐Clone CorP.), Bcl‐2 (1:50, PAA778Ra01, Cloud‐Clone CorP.), IL‐1β (1:50, PAA563Ra01, Cloud‐Clone CorP.), and IL‐18 (1:50, PAA064Ra01, Cloud‐Clone CorP.). Slices were then stained with secondary antibodies for 50 min, and diaminobenzidine was used for color development. The photographs were taken using a microscope.

2.9. Enzyme‐linked immunosorbent assay

The Nanjing Jiancheng Biological Engineering Research Institute (Nanjing, China) provided all of the enzyme‐linked immunosorbent assay kits. After 75 min of reperfusion, the solution from the coronary artery was collected, and the levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were determined in accordance with the instructions on the corresponding kit.

2.10. Quantitative real‐time PCR

Total RNA extraction experiments were carried out using TRIzol® reagent purchased from Takara Biotechnology (Dalian, China). Then, the total RNA was quantified using an ultraviolet spectrophotometer. When absorbance A260/A280 = 1.8 ~ 2.0, the total RNA could be used as a template for single‐stranded cDNA synthesis by reverse transcription using the First Strand cDNA Synthesis Kit (Thermo Scientific, #K1622). Quantitative PCR (qPCR) was then carried out using FastStart™ PCR Master (Roche, 4710436001), and the relative expressions of IL‐18 and IL‐Iβ were calculated using the 2−∆∆Ct method. GAPDH was used as an internal control. The related primer sequences were as follows: IL‐18 (F) 5′‐ATG CCT GAT ATC GAC CGA AC‐3′, (R) 5′‐TGG CAC ACG TTT CTG AAA GA‐3′; IL‐1β (F) 5′‐ACA GCA ATG GTC GGG ACA TA‐3′, (R) 5′‐TGA GAG ACC TGA CTT GGC AG‐3′; NLRP3 (F) 5′‐CAG CCA GAG TGG AAT GAT GC‐3′, (R) 5′‐GGG TGT AGC GTC TGT TGA G‐3′; ASC (F) 5′‐GGC ACA GCC AGA ACA GAA‐3′, (R) 5′‐GCA TCC AGC AAA CCA TCA‐3′; Caspase‐1 (F) 5′‐GCT TCA GTC AGG TCC ATC‐3′, (R) 5′‐ACC ACT CGG TCC AGG AAA TGC‐3′; and GAPDH (F) 5′‐CAA GTT CAA CGG CAC AGT CAA G‐3′, (R) 5′‐ACA TAC TCA GCA CCA GCA TCA C‐3′.

2.11. Western blot assay

The whole myocardial tissues were cut into pieces on ice and placed in pre‐chilled RIPA buffer cracking fluid (containing 1% PMSF) for complete homogenization. The lysate was centrifuged to collect the cell supernatants. The BCA kit (Beyotime, Shanghai, China) was used to quantify the total protein concentration. Equal amounts of protein (50 μg) were separated by 12% SDS‐PAGE electrophoresis and then transferred to a PDVF membrane (Millipore). The membranes were then treated with primary antibodies, including NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab180799, Abcam), activated‐Caspase‐1 (1:1000, PAB592Mu01, Cloud‐Clone CorP.), activated‐Caspase‐3 (1:1000, PAA626Ra01, Cloud‐Clone CorP.), Bax (1:1000, PAB343Ra01, Cloud‐Clone CorP.), Bcl‐2 (1:1000, PAA778Ra01, Cloud‐Clone CorP.), p‐Akt (1:1000, ab38449, Abcam), Akt (1:10000; ab179463, Abcam), p‐GSK3β (1:500, ab75745, Abcam), GSK3β (1:1000, ab131356, Abcam), IL‐1β (1:1000, PAA563Ra01, Cloud‐Clone CorP.) and IL‐18 (1:1000, PAA064Ra01, Cloud‐Clone CorP.) and GAPDH (1:2500, PAB932Hu01, Cloud‐Clone CorP.) for 24 h at 4°C. After washing, the membranes were incubated with secondary antibodies (1:5000, ab6734, Abcam). The bands were visualized using an ECL kit (Thermo Scientific), and Image J software was used to quantify and compare band density.

2.12. Experimental data analysis

SPSS20.0 software was used for statistical analysis of the data. The measurement data were expressed as mean ± standard deviation. One‐way ANOVA followed by Tukey's post hoc test was used to compare data between multiple groups. p < 0.05 was considered statistically significant.

3. RESULTS

3.1. TSAE inhibited MIRI‐induced activation of the NLRP3 inflammasome

Previous studies reported that TSAE played an important protective role in cardiac function and myocardial injury. 15 , 22 In this section, we investigate the effect of TSAE on NLRP3 inflammasome activation in the MIRI model. MIRI rats were administered either TSAE or a specific inhibitor targeting the NLRP3 inflammasome (MCC950), as shown in Figure 1A. Hemodynamic monitoring revealed that while malignant arrhythmias, left ventricular contraction, and diastolic function decreased in the I/R group during reperfusion and were improved by TSAE or MCC950, hemodynamics remained stable in the sham group without significant fluctuations (Figure 1B). Following TTC staining, the hearts of rats in the I/R group had gray infarct areas compared to those in the sham group, and treatment with TSAE or MCC950 reduced the infarct area (Figure 1C). Based on these findings, it could be concluded that the myocardial I/R model was successfully constructed. IL‐18 and IL‐1β are important inflammatory factors that operate downstream of the NLRP3 pathway. Using qPCR and immunohistochemical staining, the expressions of IL‐18 and IL‐1β were significantly increased following I/R stimulation, which was significantly alleviated by TSAE or MCC950 reperfusion (Figure 1D,E). Changes in the expression of NLRP3 inflammasome‐related markers were then evaluated, and the results revealed that ASC, NLRP3, and Caspase‐1 levels in the I/R group were highly elevated; however, all of these levels were decreased by TSAE or MCC950 treatment (Figure 1F,G). In conclusion, these experimental results indicated that TSAE could inhibit NLRP3 inflammasome activation in MIRI.

3.2. TSAE alleviated I/R‐induced myocardial damage by inhibiting the activation of NLRP3 inflammasome

After cardiac reperfusion, myocardial pathological changes were evaluated by Hematoxylin & eosin (H&E) staining. As shown in Figure 2A, there was no degeneration, necrosis, or inflammatory cell infiltration in rats in the sham group, and myocardial cells were distributed in an orderly manner. In contrast, myocardial cells in the I/R group were disordered and had significant levels of cell degeneration, necrosis, and inflammatory cell infiltration. Importantly, this phenomenon was significantly mitigated by the administration of TSAE or MCC950. In addition, this study also investigated whether TSAE treatment affected cardiac left ventricular function by modulating the NLRP3 inflammasome. The results in Figure 2B indicate that I/R stimulation significantly reduced LVDP and ±dp/dt max but had no discernible effect on HR. Notably, the inhibitory effect of I/R on LVDP and ±dp/dt max was significantly reversed by administration of TSAE or MCC950. Similar trends were observed in myocardial cell oxidative damage and apoptosis as well. CK, LDH, and MDA concentrations, as well as the pro‐apoptotic‐related marker (Caspase‐3, Bax) protein levels, increased significantly, while SOD content and Bcl‐2 protein levels decreased after I/R stimulation, all of which were abolished by TSAE or MCC950 treatment (Figure 2C,D). Therefore, in combination with the results in Figure 1, it was speculated that the protective effect of TSAE on cardiac function and myocardial injury was correlated with NLRP3 inflammasome inactivation.

FIGURE 2.

FIGURE 2

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TSAE alleviated I/R‐induced myocardial injury by inhibiting the activation of the NLRP3 inflammasome. Animals were treated and grouped as shown in Figure 1. (A) Pathological changes in myocardial tissues detected by H&E staining. (B) Hemodynamic detection of heart rate (HR), left ventricular development pressure, and ±dp/dt max. (C) Detection of creatine kinase (CK), malondialdehyde, lactate dehydrogenase, and superoxide dismutase levels using corresponding detection kits. (D) Western blot assay for determination of protein levels of apoptosis‐related markers, including Caspase‐3, Bax, and Bcl‐2. Data were expressed as mean ± standard deviation. N = 12 per group. *p < 0.05; **p < 0.01; and ***p < 0.001.TSAE, total saponins of Aralia elata (Miq.) Seem.

3.3. TSAE promoted Akt and GSK‐3β phosphorylation in the MIRI model

The relationship between TSAE and PI3K/Akt signaling in the rat MIRI model was explored. MIRI rats were treated with either TSAE or LY29004 alone or in combination, as shown in Figure 3A. The activation of proteins involved in PI3K/Akt signaling was then assessed by western blot assay, which revealed that phosphorylated Akt and GSK3β levels were elevated in myocardial tissues of I/R‐induced rats. Importantly, TSAE treatment significantly enhanced phosphorylated Akt and GSK3β levels compared to the I/R group, but co‐administration of LY29004 diminished these promoting effects (Figure 3B,C), indicating that TSAE activates PI3K/Akt signaling in MIRI.

3.4. Inhibition of PI3K/Akt signaling partially limited the protective role of TSAE in I/R‐induced myocardial injury

The next objective was to investigate the impact of PI3K/Akt signaling on the protective role of TSAE in MIRI rats. Following H&E staining, TSAE treatment reduced myocardial cell swelling, and cell degeneration, necrosis, and inflammatory cell infiltration also decreased in comparison to the I/R group, while co‐treatment with LY290004 further increased myocardial cell necrosis and inflammatory cell infiltration, clearly inhibiting the biological effects of TSAE (Figure 4A). Consistently, TSAE treatment increased LVDP and ±dp/dt max levels while having no discernible difference in HR relative to the I/R group, but these effects were impeded by LY290004 (Figure 4B). Additionally, treatment with LY290004 reversed the stimulatory effects of TSAE on SOD activity and the inhibitory effects on CK, LDH, and MDA levels (Figure 4C). Immunohistochemical staining revealed that LY290004 treatment significantly increased Caspase‐3 and Bax levels but downregulated Bcl‐2 levels, which diminished the roles of TSAE (Figure 4D). These findings suggest that PI3K/Akt signaling is involved in the protective processes of TSAE in I/R‐induced myocardial injury.

FIGURE 4.

FIGURE 4

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Inhibition of PI3K/Akt signaling partly restrained the protective roles of TSAE in I/R‐induced myocardial injury. Animals were treated and grouped as shown in Figure 3. (A) Pathological changes in myocardial tissue detected by H&E staining. (B) Hemodynamic measurement of the changes of heart rate (HR), left ventricular development pressure, and ±dp/dt max. (C) Evaluation of creatine kinase (CK), malondialdehyde, lactate dehydrogenase, and superoxide dismutase levels using corresponding detection kits. (D) Immunohistochemical staining for analysis of protein levels of apoptosis‐related markers, including Caspase‐3, Bax, and Bcl‐2. Data were expressed as mean ± standard deviation. N = 12 per group. TSAE, total saponins of Aralia elata (Miq.) Seem. *p < 0.05; **p < 0.01; and ***p < 0.001.

3.5. Inactivation of PI3K/Akt signaling abolished the modulation of TSAE on NLRP3 inflammasome

Previous studies have shown that Akt plays a crucial regulating role in the downstream NLRP3 inflammasome activation. 23 , 24 In this study, the correlation between TSAE, PI3K/Akt, and NLRP3 inflammasome was investigated. qPCR and western blot assays revealed that IL‐18 and IL‐1β mRNA and protein levels were markedly increased in I/R‐induced myocardial tissues of rats, which were partially decreased by TSAE reperfusion. However, following LY290004 treatment, the suppressive effects of TSAE reperfusion were almost abolished (Figure 5A,B). Additionally, the data in Figure 5C,D showed that co‐treatment with LY290004 significantly reduced the inhibitory effect of TSAE on NLRP3, ASC, and Caspase‐1 expression in the MIRI rat model, indicating that PI3K/Akt signaling is a crucial pathway for TSAE to regulate NLRP3 inflammasome.

FIGURE 5.

FIGURE 5

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Inactivation of PI3K/Akt signalling abolished the modulation of total saponins of Aralia elata (Miq.) Seem (TSAE) on the NLRP3 inflammasome. Animals were treated and grouped as shown in Figure 3. (A‐B) Quantitative PCR (qPCR) and western blot assays for quantification of IL‐18 and IL‐1β mRNA and protein levels. (C‐D) qPCR and western blot assays for evaluating changes in expression of NLRP3 inflammasome‐associated markers, including NLRP3, ASC, and Caspase‐1. All data were indicated as mean ± standard deviation. N = 12 per group. *p < 0.05; **p < 0.01; and ***p < 0.001.

4. DISCUSSION

AMI is one of the most common fatal diseases in the world, with an increased incidence among young people. 25 In recent years, improvement of MIRI after surgery or thrombolytic therapy has become an important topic in cardiovascular clinical and scientific research. The pathogenesis of MIRI is complex and involves several factors, including oxidative stress, inflammation, and mitochondrial dysfunction. 26 , 27 , 28 Previous studies provided a direction for MIRI treatment by demonstrating that pre‐ or post‐treatment with drugs such as Bauhinia championii flavone and vitexin can minimize cell necrosis, decrease infarct size, and enhance cardiac function. 28 , 29 In the present study, an isolated myocardial I/R rat model was developed using the Langendroff instrument, and the results revealed that administration of TSAE significantly alleviated NLRP3 inflammasome‐mediated MIRI by activating the PI3K/Akt pathway.

AS is a widely used edible homologous plant with a high medicinal value. Pharmacological studies indicated that saponins and polysaccharides extracted from AS exhibit cardiovascular protection, anti‐tumor, liver protection, anti‐oxidant stress, and anti‐inflammatory properties. According to Cheng et al., saponin formation in AS can be regulated by sunlight and stress levels, and the amount of saponin showed a significant correlation with the activity of anti‐oxidant enzymes, 30 suggesting that increased saponins may be a host's protective response to oxidative stress. In atherosclerosis, TSAE treatment reduced atherosclerotic plaque size and apoptotic level of the aortic valve in high‐fat diet‐induced ApoE−/− mice and promoted endothelial cell survival by raising mitochondrial membrane potential. 31 Similar protective roles were observed in a study by Zhou et al. 18 In addition, TSAE treatment decreased myocardial infarct area, LDH, CK, and MDA levels and increased the activities of Ca2+–Mg2+‐ATPase, calcineurin phosphatase, Na+–K+‐ATPase, SOD, and sarcoplasmic reticulum Ca2+‐ATPases, 15 illustrating the protective effect of TSAE on I/R‐induced injury. Similarly, our experimental data showed that TSAE treatment increased LVDP, ±dp/dt max, SOD activity, and Bcl‐2 levels but decreased CK, MDA, and LDH levels, Caspase‐3 and Bax expression, production of inflammatory mediators (IL‐18, IL‐1β), as well as NLRP3‐inflammasome formation in I/R‐induced myocardial tissues. These results indicated that TSAE treatment reduced myocardial enzyme leakage, improved left ventricular systolic and diastolic function, and alleviated myocardial damage after I/R stimulation. Notably, all of these TSAE‐mediated changes were similar to MCC950 treatment, suggesting that protective mechanisms of TSAE may be associated with reduced NLRP3 inflammasome‐mediated inflammatory responses.

In the current study, it was observed that TSAE administration clearly activated PI3K/Akt signaling in I/R‐stimulated myocardial tissue and that LY29004 treatment impaired the effect of TAST on systolic and diastolic function and anti‐oxidant capacity. A similar regulatory role of TSAE on PI3K/Akt signaling was reported by Zhou et al. 18 Recently, TASE has been established to have important anti‐inflammatory and anti‐oxidant properties. 15 Activation of the PI3K/Akt pathway was influenced by a large number of inflammatory/oxidative stress‐related factors. For example, TNF‐α can inactivate PI3K/Akt signaling by enhancing PTEN expression, leading to high‐fat diet‐induced insulin resistance. 32 Bupivacaine suppressed angiogenesis by inhibiting Akt/mTOR and activating AMPK in an oxidative stress‐dependent manner. 33 HO‐1 can protect the nerve of rats with cerebral hemorrhage by regulating PI3K/Akt signaling pathway. 34 This might be a possible reason why TSAE regulates the PI3K/Akt pathway. However, the exact mechanisms remain to be elucidated.

Interestingly, recent research has confirmed that PI3K/Akt signaling is responsible for the regulation of NLRP3 inflammasome formation and associated pyroptosis activation. Curcumin was found to rescue doxorubicin‐stimulated cardiomyocyte pyroptosis by reducing NLRP3, Caspase‐1, and IL‐18 levels, which are dependent on PI3K/Akt/mTOR pathway regulation. 35 A study by Zhuo et al. also confirmed that LSD1 silencing repressed Ox‐LDL‐triggered NLRP3 activation and inflammation in RAW264.7 cells by modulating PI3K/Akt/mTOR pathway‐mediated autophagy. 36 According to research by Liu et al., the phosphorylation of AMPK and GSK‐3β inhibited NLRP3 inflammasome activation in astrocytes. 37 Similarly, activation of Akt/GSK‐3β suppressed NLRP3 inflammasome formation, effectively protecting cardiomyocytes or microglial cells from I/R injury. 38 , 39 These findings suggested a possible connection between TSAE, PI3K/Akt signaling, and the NLRP3 inflammasome. Consistently, our experimental findings showed that LY29004 partly attenuated the inhibitory effect of TSAE on NLRP3 inflammasome‐related markers (NLRP3, ASC, Caspase‐1) and IL‐18 and IL‐1β production. Based on the above findings, it was concluded that TSAE reduced NLRP3 inflammasome activity by activating the PI3K/Akt signaling pathway, thereby reducing I/R‐induced oxidative stress response and restoring cardiac function.

5. CONCLUSION

In conclusion, post‐treatment with TSAE alleviated I/R‐induced myocardial damage in isolated rat hearts, which is associated with attenuated oxidative stress response and apoptosis, enhanced anti‐oxidant capacities of the heart, and decreased inflammatory response. The suppression of NLRP3 inflammasome by PI3K/Akt signaling may be partially related to the protective mechanism of TSAE. In addition, all experimental results can provide a theoretical basis for AMI treatment and prevention.

CONFLICTS OF 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.

ACKNOWLEDGMENT

We would like to thank the anonymous reviewers who have helped to improve the paper.

Sun L, Lu W‐X, Li H, Feng D‐Y, Nie J‐X. Total saponins of Aralia elata (Miq.) Seem. alleviate myocardial ischemia‐reperfusion injury by promoting NLRP3‐inflammasome inactivation via PI3K/Akt signaling. Kaohsiung J Med Sci. 2023;39(3):290–301. 10.1002/kjm2.12627

Funding information Beijing Municipal Natural Science Foundation, Grant/Award Number: 7172124; National Natural Science Foundation of China, Grant/Award Number: General Program: 81273691; the initial fund for new teachers of Beijing University of Chinese Medicine, Grant/Award Number: 2021‐JYB‐XJSJJ067

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