Suppressing receptor-interacting protein 140: a new sight for esculetin to treat myocardial ischemia/reperfusion injury

Abstract

The purpose of the present study was to evaluate the cardioprotective effect of esculetin (ES) on myocardial ischemia/reperfusion (I/R) damage in rats and investigate the potential mechanism. Rats were randomly assigned to 4 groups, namely, sham group, I/R group, I/R + ES (20 mg kg⁻¹, intragastric administration, 7 days) group, and I/R + ES (40 mg kg⁻¹, intragastric administration, 7 days) group. The indices of myocardial injury, inflammatory cytokines and apoptosis-related parameters were detected. ST segment elevation of electrocardiograph (ECG) was observed in ischemia rats. The results demonstrated that ES administration effectively decreased the levels of creatinine kinase (CK), lactate dehydrogenase (LDH), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) in serum and reduced the myocardial infarction area compared with the I/R group. ES treatment also markedly restored the activity of superoxide dismutase (SOD) and decreased the amount of malondialdehyde (MDA) of the I/R rats. Moreover, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay suggested that ES treatment suppressed myocardial cell apoptosis, which might be associated with the up-regulation of Bcl-2 and the down-regulation of Bax, caspase-3 and caspase-9. In addition, ES attenuated myocardial I/R induced injury in rats by inhibiting the receptor-interacting protein 140 (RIP140)/nuclear factor kappa B (NF-κB) inflammatory pathway. Collectively, it could be assumed that ES significantly improved myocardial I/R injury in rats partially through suppression of myocardial apoptosis and inflammation mediated by the RIP140/NF-κB pathway.

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Heart disease is one of the major pervasive diseases around the world. Extensive investigators have researched the cardioprotective therapy during the last decade.^1,2 Notably, myocardial ischemia/reperfusion (I/R) attracts considerable attention due to its complex pathogenesis that early reperfusion is critical for tissue salvage, whereas excessive reperfusion may result in additional myocardial injury beyond that produced by ischemia alone.³ IR contributes to serious acute or chronic myocardial damage such as myocardial ultrastructural alteration, remodeling, systolic and diastolic dysfunction.⁴ Therefore, there is an urgent need to elucidate the underlying mechanism responsible for I/R injury and seek novel therapeutic strategies for treating ischemic–reperfused heart damage.

Inflammatory reactions are closely associated with the physiological process of myocardial ischemia reperfusion injury.⁵ I/R injury is attributed to the overproduction of various pro-inflammatory cytokines, including TNF-α and IL-6.⁶ It is acknowledged that RIP140 plays an important role in the mediation of inflammatory cascades.⁷ RIP140, which can activate the pro-inflammatory cytokine generation to regulate the inflammatory progression in macrophages, is reported to interact with the essential transcriptional molecule, i.e., NF-κB or cAMP response element binding protein (CREB), to modulate the expressions of pro-inflammatory cytokines IL-6 and TNF-α.^8,9 RIP140 is demonstrated to regulate the production of NF-κB dependent pro-inflammatory cytokines under an experimental model of endotoxin tolerance.¹⁰

There has been much interest in studying the strict correlation between cardiomyocyte apoptosis and heart diseases, including I/R damage.¹¹ Apoptosis was regarded as a type of genetically regulated programmed cell death to avoid the indiscriminate catastrophe during I/R injury. Evidence revealed that cardiomyocyte apoptosis initiated after ischemia and was amplified by a reperfusion procedure.¹² Bcl-2 and cysteine protease family are considered to be responsible for I/R-induced apoptosis. Previous studies proposed that the negative and positive interactions among the Bcl-2 family members played crucial roles in regulating the cardiomyocyte apoptotic progress.^13,14 Thus, blocking the apoptotic pathway may minimize cardiac injury and reduce the occurrence of ischemia myocardial.¹⁵

In recent years, a variety of natural products have been used for disease intervention.^16,17 Esculetin (ES, 6,7-dihydroxycoumarin) is a derivative of coumarin, which is used as a traditional Chinese medicine. Coumarin is reported to possess a diversity of biological and pharmacological activities such as anti-oxidative, anti-viral, anti-tumor and anti-proliferative effects.¹⁸ It has been documented that ES inhibits lipoxygenase and exerts cytoprotective effects against oxidative stress-induced dysfunction.¹⁹ In addition, some investigators confirmed that ES exerted positive effects on lipopolysaccharide-stimulated acute lung injury.²⁰ However, there has been no report with respect to the therapeutic effect of ES on I/R injury. Therefore, the present study was designed to identify the cardioprotective effect of ES on I/R injury in rats and explore its potential mechanism.

Methods

Reagents

ES (purity > 99%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for the determination of IL-6 and TNF-α were produced by Nanjing KeyGEN Biotech. CO., LTD. (Nanjing, China). Primary antibodies against Bax (1 : 1000), Bcl-2 (1 : 1000), caspase-3 (1 : 1000), caspase-9 (1 : 1000), NF-κB (1 : 1000), p-NF-κB (1 : 1000), IκBα (1 : 1000) and p-IκBα (1 : 1000) were provided by Cell Signaling Technology Inc (Beverly, MA, USA). Primary antibodies against RIP140 (1 : 500) were purchased from Santa Cruz Biotechnology (CA, USA).

Animals

Male Sprague Dawley rats (280 ± 20 g) were obtained from Jiangning Qinglongshan Animal Cultivation Farm (Nanjing, China) and housed at a constant temperature and relative humidity under a regular 12 h light/dark cycle. Animals were supplied with standard chow and water ad libitum.

Ethical statement

All the experimental procedures were performed in accordance with the Nanjing University of Chinese Medicine for the Care and Use of Laboratory Animals. The experimental procedures were approved by the Nanjing University of Chinese Medicine. All the experimental procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Experimental protocol and drug administration

The surgical protocol was conducted as previously described by Bhindi et al.²¹ The success of the myocardial ischemia/reperfusion was confirmed by regional cyanosis of the myocardium and a typical ECG measurement.²² The slipknot was loosened after 30 min of ischemia and the myocardium was reperfused for 24 h. At the end of the experimental period, the animals were anesthetized with chloral hydrate (3.5 ml kg⁻¹) intraperitoneally. Then, the rats were sacrificed and blood samples were collected for biochemical assays. The hearts were harvested for morphological, biochemical and molecular studies. Taking accidental deaths caused by anesthesia or failed surgery into consideration, the number of rats in each group was as follows: sham group (n = 10), I/R group (n = 8), I/R + ES (20 mg kg⁻¹, intragastric administration, 7 days) group (n = 7), I/R + ES (40 mg kg⁻¹, intragastric administration, 7 days) group (n = 9).

Electrocardiogram (ECG) measurement

ECG was monitored using the BL-420S Biologic Function Experiment system (Chengdu, China). Ischemia was verified by ST segment elevation recorded by ECG. The results were expressed by taking sham as the control.

Determination of myocardial infarct size

After reperfusion, the hearts of the animals were excised and kept at −20 °C for 15 min. Hearts were cut into 5 mm slices, incubated with 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution for 15 min in the dark at 37 °C. TTC stained area (red staining, ischemic area) and non TTC stained areas (white, infarct area) were analyzed using the Image-Pro Plus image analysis software (Media Cybernetics, LP, USA). Finally, the ratio of myocardial infarct area (infarct area/area at risk%, INF/AAR%) was calculated.

Determination of cardiac marker enzymes

Myocardial cellular damage was detected by measuring the serum LDH and CK-MB. Serum CK-MB and LDH activities were assayed spectrophotometrically according to the manufacturer's instructions of commercial kits.

Determination of antioxidant system and lipid peroxidation products

SOD activity and MDA level in the serum were measured using assay kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All measurements were performed according to the manufacturer's instructions.

Determination of inflammatory cytokines in serum

The contents of IL-6 and TNF-α in serum were measured using ELISA kits according to the manufacturer's instructions. The cytokines concentrations were quantified by reference to standard curves. The optical density (OD) of each well was read at 450 nm.

Histopathological heart analysis

At the end of the experiment, the hearts were collected and fixed in 10% formalin solution. The heart tissue was dehydrated in graded alcohol, embedded in paraffin wax and stained with hematoxylin and eosin (H&E). After staining, the pathological changes were observed by light microscopy. A yellow arrow indicated edema, a green arrow indicated inflammatory infiltration and a blue arrow indicated cardiac necrosis. The bar graph reflects the scores of edema, cardiac necrosis and inflammatory infiltration in the infarcted regions. Moreover, the pathological alterations were scored in accordance with the previous study.²³ In general, the severity of the heart was graded on a scale of 0.5 to 4: 0.5 = minor, 1 = mild, 2 = moderate, 3 = severe, and 4 = very severe.

Determination of myocardial apoptosis

The TUNEL System (Promega) was carried out according to the manufacturer's protocol. Heart tissues were fixed with 4% neutral formalin in PBS for 25 min at 4 °C. After washing with PBS twice, the tissues were kept in 70% ethanol at −20 °C overnight. Then, the samples were subsequently saturated and permeabilized with 0.2% Triton X-100 for 5 min. Next, 100 μl buffer containing 45 μl equilibration buffer, 5 μl nucleotide mix and 1 μl rTdT enzyme was treated for 10 min. After washing for 15 min with SSC and PBS, the samples were incubated for DAPI staining at room temperature in the dark for 15 min. Acquisition of the images was performed under a fluorescence microscope. TUNEL positive (brown) cardiac myocytes were regarded as apoptotic cells and the TUNEL positive percentage was calculated from the number of TUNEL-positive cells divided by the total number of cells by an observer who was blind to the experiment.

Western blot

Heart tissues were chopped into small pieces and homogenized in ice-cold RIPA buffer (Beyotime, Nanjing, China). Protein concentrations were determined using a BCA protein assay (Beyotime, Nanjing, China). Thereafter, protein extracts were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk in Tris buffer saline and incubated with corresponding primary antibodies at 4 °C overnight. After washing with Tris buffered saline-Tween 20 (TBST), the membranes were incubated with a horseradish peroxidase conjugated secondary antibody for 1.5 h at room temperature. Thereafter, the antibody-reactive bands were visualized using an ECL Key-GEN system (KeyGEN Biotechnology, Nanjing, China).

Statistical analysis

All data were normally distributed and were presented as mean ± SDs. Results were analyzed by one-way ANOVA with Tukey multiple comparison test. The values were regarded as statistically significantly different at P < 0.05.

Results

Effects of ES on ST-segment elevation

Electrocardiographic patterns of normal and experimental rats are revealed in Fig. 1. The ST-segment was dramatically elevated and R-amplitude was decreased in I/R rats compared with those in the sham group. The results represented that I/R damage model was well established. ES obviously ameliorated the above described conditions at the 20 and 40 mg kg⁻¹ doses, which partially shows the protective effects.

Fig. 1 Effects of esculetin (ES) on ST-segment elevation. The rats were intragastrically administered with ES (20, 40 mg kg⁻¹). 1 h later, the animals received 30 min ischemia and were then reperfused for 24 h. The number of rats in each group was as follows: sham (n = 10), ischemia/reperfusion (I/R) (n = 8), I/R + ES 20 mg kg⁻¹ (n = 7), I/R + ES 40 mg kg⁻¹ (n = 9). Values are expressed as mean ± SDs. (A) Sham, (B) I/R, (C) I/R + ES (20 mg kg⁻¹), (D) I/R + ES (40 mg kg⁻¹). Compared with sham: ##P < 0.01; compared with model: **P < 0.01.

Effects of ES on myocardial infarction

The infarct size of individual animal was evaluated by TTC staining. There was no infarct damage in the sham group of rats. The percent of infarct size in I/R rats was remarkably higher than those in the sham group. In contrast, the myocardial infarction area in ES treated group was effectively reduced compared with the I/R group. These results clearly suggested the protective effect of ES treatment on myocardial I/R injury (Fig. 2).

Fig. 2 Esculetin (ES) reduced the myocardial infarction area induced by ischemia/reperfusion (I/R). (A) Representative 2,3,5-triphenyltetrazolium chloride (TTC) staining of samples from rats subjected to different treatment groups. (B) The ratio of myocardial infarction area (INF) to area at risk (AAR). The rats were intragastrically administered with ES (20, 40 mg kg⁻¹). 1 h later, the animals received 30 min ischemia and were then reperfused for 24 h. The number of rats in each group was as follows: sham (n = 10), I/R (n = 8), I/R + ES 20 mg kg⁻¹ (n = 7), I/R + ES 40 mg kg⁻¹ (n = 9). Values are expressed as mean ± SDs. (A) Sham, (B) I/R, (C) I/R + ES (20 mg kg⁻¹), (D) I/R + ES (40 mg kg⁻¹). Compared with sham: #P < 0.05, ##P < 0.01; compared with model: *P < 0.05, **P < 0.01.

Effects of ES on cardiac marker enzymes

To detect myocardial injury marker enzymes, the levels of CK-MB and LDH in serum were measured. As depicted in Fig. 3, the activities of CK-MB and LDH were obviously increased in I/R animals compared with those in sham rats. On the contrary, ES treatment (40 mg kg⁻¹) significantly decreased CK-MB and LDH vitalities compared with the I/R group, particularly in those with higher ES doses.

Fig. 3 Effects of esculetin (ES) on the levels of lactate dehydrogenase (LDH), creatinine kinase (CK), superoxide dismutase (SOD) and malondialdehyde (MDA) in serum. The rats were intragastrically administered with ES (20, 40 mg kg⁻¹). 1 h later, the animals received 30 min ischemia and were then reperfused for 24 h. The number of rats in each group was as follows: sham (n = 10), I/R (n = 8), I/R + ES 20 mg kg⁻¹ (n = 7), I/R + ES 40 mg kg⁻¹ (n = 9). Values are expressed as mean ± SDs. Compared with sham: ##P < 0.01; compared with model: *P < 0.05, **P < 0.01.

Effects of ES on antioxidant systems and lipid peroxidation

To estimate lipid peroxidation and endogenous anti-peroxidative enzymes, the levels of MDA and SOD in serum were detected. The significant increased MDA content and obviously decreased SOD activity in the serum was observed in the I/R rats compared with the sham rats. It is worth noting that ES administration attenuated these situations in contrast with those in the I/R group. It can also be noted that for SOD activity, ES (40 mg kg⁻¹) exerted much greater protective efficiency than ES (20 mg kg⁻¹). The obtained data suggested that ES evidently enhanced the activities of the enzymatic antioxidant defense system (Fig. 3).

Effects of ES on the levels of inflammatory cytokines in serum

Inflammatory reaction is one of the major features in myocardial infarction. To determine whether ES could inhibit the inflammatory responses during myocardial I/R injury, the serum levels of TNF-α and IL-6 were assessed. It was proven that there were significant increases of serum TNF-α and IL-6 in the I/R group. The serum TNF-α and IL-6 contents were effectively decreased in the ES (40 mg kg⁻¹)-treated group compared with those in the I/R group rats, which were statistically better than those of ES (20 mg kg⁻¹). Our results indicated that ES reduced the inflammatory cytokine contents in the serum of the I/R injury rats (Fig. 4).

Fig. 4 Effects of esculetin (ES) on the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum. The rats were intragastrically administered with ES (20, 40 mg kg⁻¹). 1 h later, animals received 30 min ischemia and were then reperfused for 24 h. The number of rats in each group was as follows: sham (n = 10), I/R (n = 8), I/R + ES 20 mg kg⁻¹ (n = 7), I/R + ES 40 mg kg⁻¹ (n = 9). Values are expressed as mean ± SDs. Compared with sham: ##P < 0.01; compared with model: *P < 0.05, **P < 0.01.

Effects of ES on histopathological lesions

Hematoxylin and eosin (H&E) staining was conducted to evaluate the protective role of ES on physiological impairment. As illustrated in Fig. 5, a normal myofibrillar structure with striations, branched appearance and continuity adjacent myofibrils was observed with the sham group, whereas the tissues from the I/R rats revealed disordered myocardial structure, edema, inflammatory infiltration and cardiac necrosis compared with those in the sham group. Interestingly, treatments with ES (20 and 40 mg kg⁻¹) markedly inhibited edema, inflammatory infiltration and cardiac necrosis compared with those in the I/R group. The results demonstrated that ES could ameliorate the histopathology condition in myocardial tissue.

Fig. 5 Effects of esculetin (ES) on histopathological lesions (×200). The rats were intragastrically administered with ES (20, 40 mg kg⁻¹). 1 h later, animals received 30 min ischemia and then reperfused for 24 h (n = 3). Sham group showed normal cardiac fibers without any edema, inflammatory infiltration or cardiac necrosis. I/R group showed myocardial structure disorder, edema, inflammatory infiltration and cardiac necrosis. ES (20 mg kg⁻¹) group showed less inflammatory infiltration and edema. Treatment with ES (40 mg kg⁻¹) obviously attenuated all these alterations compared with those in the I/R group. The yellow arrow indicates edema, the green arrow indicates inflammatory infiltration, and the blue arrow indicates cardiac necrosis. The bar graph reflects the scores of edema, cardiac necrosis and inflammatory infiltration in the infarcted regions.

ES reduced cardiomyocyte apoptosis induced by I/R

It was widely acknowledged that apoptosis contributes to myocardial cell death. To examine the effect of ES on myocardial cell apoptosis, TUNEL staining was performed. The TUNEL-positive brown colored cells were considered as apoptotic cells. As shown in Fig. 6, almost no TUNEL-positive cells were observed in the sham group. Notably, a significant increased number of TUNEL-positive cells were detected in myocardial tissue from hearts subjected to I/R. Herein, administration of ES (40 mg kg⁻¹) exerted a significant anti-apoptotic effect as shown by the reduced number of TUNEL-positive cells, which was a slightly more potent than treatment with ES (20 mg kg⁻¹). The results suggested that ES could inhibit the cardiac cell apoptosis of myocardial I/R injury in rats.

Fig. 6 Esculetin (ES) reduced cardiomyocyte apoptosis induced by I/R. The rats were intragastrically administered with ES (20, 40 mg kg⁻¹). 1 h later, the animals received 30 min ischemia and were then reperfused for 24 h (n = 3). The green arrow indicates TUNEL positive cells.

Effects of ES on cardiomyocyte apoptosis-related proteins

To further investigate the anti-apoptotic mechanism of ES in inhibiting cardiac ischemic/reperfusion injury, the expressions of apoptosis-related proteins were assayed. Compared with the sham group, the levels of Bax, caspase-3, cleaved caspase-3 and caspase-9 were increased, whereas the Bcl-2 level was decreased. Treatment with ES significantly down-regulated the expressions of Bax, caspase-3, cleaved caspase-3 and caspase-9, whereas the expression of Bcl-2 was up-regulated. Furthermore, the effect of ES at a higher dose was more pronounced than that of lower dose on Bax and Bcl-2 (Fig. 7).

Fig. 7 Effects of esculetin (ES) on cardiomyocyte apoptosis-related proteins. The rats were intragastrically administered with ES (20, 40 mg kg⁻¹). 1 h later, the animals received 30 min ischemia and were then reperfused for 24 h. Values were expressed as mean ± SDs. Compared with sham: ##P < 0.01; compared with model: *P < 0.05, **P < 0.01. (n = 3).

Effects of ES on RIP140/NF-κB pathway

To further explain the mechanism behind cardioprotection of ES, the expressions of RIP140/NF-κB signaling-related proteins were detected. Compared with the sham group, the expressions of RIP140, p-IκBα, IκBα, p-NF-κB and NF-κB were up-regulated. As expected, ES treatment notably down-regulated the expressions of proteins related to the RIP140/NF-κB pathway, taking the expression of GAPDH as an inner control. Treatment with ES (40 mg kg⁻¹) was slightly more significant for IκBα phosphorylation than that in the ES (20 mg kg⁻¹)-treated rats (Fig. 8).

Fig. 8 Effects of esculetin (ES) on the RIP140/NF-κB pathway. The rats were intragastrically administered with ES (20, 40 mg kg⁻¹). 1 h later, the animals received 30 min ischemia and were then reperfused for 24 h. Values are expressed as mean ± SDs. Compared with sham: ##P < 0.01; compared with model: *P < 0.05, **P < 0.01. (n = 3).

Discussion

In the present study, ES has been demonstrated to alleviate the cardiac ischemic/reperfusion injury in rats. I/R model was established and verified by the loss of myocardial membrane integrity with histological observation, ST segment elevation and larger infarct size. Encouragingly, the experiment results displayed that ES treatment exerted considerable cardioprotective effects. In addition, administration with ES also decreased the serum levels of CK-MB, LDH and inflammatory cytokines, as well as lipid peroxidation. ES also significantly suppressed cardiomyocyte apoptosis and attenuated the expressions of proteins associated with the RIP140/NF-κB pathway.

CK-MB, mainly distributed in the myocardium, is the diagnostic marker for necrotic damage of the myocardial membrane.²⁴ LDH is the specificity myocardial enzyme in the cytoplasm and releases into the blood during myocardial ischemia.²⁵ These two types of myocardial enzymes are often considered as the criteria in myocardial ischemia–reperfusion injury. More importantly, treatment with ES reduced the levels of these enzymes. Taking the amelioration of ST segment elevation, myocardial infarction and histological condition into account, it was reasonable to speculate that ES exhibited potential cardioprotective effects against myocardial ischemia/reperfusion injury in rats.

Anti-oxidative enzymes are involved in the intracellular mechanisms against inflammatory stresses. One of the major reasons for I/R injury is the imbalance between oxidant and antioxidant defense.²⁶ The levels of SOD and MDA are the principal patho-physiological indicators for evaluating free radical metabolisms. In general, SOD activity reflects the cellular capability of scavenging free radicals.²⁷ Moreover, MDA level is widely applied for the diagnosis of lipid peroxidation.²⁸ Excessive serum MDA is an important index of severe oxidative stress.²⁹ The amelioration of SOD activity and MDA content is the effective pharmacological intervention for treating ischemia myocardial. In the current study, SOD activity was significantly decreased, whereas MDA level was remarkably increased in the heart tissues of I/R rats, which indicated a severe oxidation stress that was induced by I/R injury. ES administration dramatically increased the SOD activity and decreased the MDA level in the myocardium, which confirmed that ES could inhibit lipid peroxidation during the intervention of I/R injury.

Apoptosis is a mainstay of cardiac tissue damage of reperfusion after ischemia. TUNEL assay was performed to examine myocardial apoptosis. Treatment with ES at doses of 20 and 40 mg kg⁻¹ resulted in significant suppression of cardiomyocyte apoptosis, which indicated that the anti-apoptotic effect of ES in the early phase of reperfusion could contribute to the attenuation of later myocardial necrosis (myocardial infarction). Caspase-9 is engaged by caspase-3 and functions as a cleavage mediator of caspase-3, which is a central modulator in the execution phase of cell apoptosis.³⁰ The activation of caspase-3 is driven by a series of signaling transduction molecules, among which the interaction between pro-apoptosis Bax and anti-apoptosis Bcl-2 family play a pivotal role.³¹ Bcl-2 and Bax form a heterodimer, then prevent Bax homodimerization and lead to serious death and survival signaling.³² Our present study revealed that I/R injury resulted in Bcl-2 down-regulation, Bax, caspase-3 and caspase-9 up-regulations, which was consistent with previous researches,³³ while ES administration significantly reversed these alterations. The results clearly suggested that ES might exert its anti-apoptotic effects through apoptotic signaling.

Inflammation has been widely recognized as a major driving factor in the ischemic process.³⁴ Lots of evidence proposed that enhanced levels of inflammatory mediators were highly related to ischemia.³⁵ Pro-inflammatory cytokines, including TNF-α and IL-6, participate in the initiation and regulation of inflammatory responses.³⁶ In the current study, the inflammatory stresses induced by I/R were reflected by TNF-α and IL-6 elevation, whereas ES treatment significantly decreased the levels of serum TNF-α and IL-6, which suggested that its cardioprotective effects were possibly associated with anti-inflammatory properties.

RIP140 is a negative transcriptional coregulator interacting with several members of the nuclear receptors superfamily. Emerging evidence indicates the key role of RIP140 in cardiovascular diseases.³⁷ Previous research suggested that RIP140 exerted a regulatory effect in the pathogenesis of atherosclerosis.³⁸ RIP140 knockout (KO) and transgenic (Tg) mice overexpressed RIP140 (RIP140 Tg) showed additional roles of the protein in heart.³⁹

Accumulating evidence indicated that RIP140 was highly related to inflammatory process in cardiac dysfunction by activating NF-κB cascades and inducing the secretion of inflammatory cytokines.⁴⁰ It has been reported that there is a positive feed-back loop between NF-κB and inflammatory cytokines.⁴¹ NF-κB activation regulates the transcriptions of pro-inflammatory genes such as TNF-α and IL-6.⁴² On the other hand, the inflammatory mediators trigger the initial inflammatory responses.⁴⁴ In addition, former literatures reported that RIP140 was involved in the mediation of Bcl-2.⁴³ Kim et al. also elicited that RIP140 suppressed TR4-mediated Bcl-2 gene expression via the interaction with the TR4 AF1 domain, which consequently regulated caspase-3 activity.⁴⁴ It can be noted that previous investigators proposed that RIP140 played a critical role in the pathophysiology of cardiovascular disease by activating cardiomyocyte mitochondrial apoptosis through the activation of NF-κB.⁴⁵ Our study showed the activation of RIP140/NF-κB pathway during I/R injury. Notably, ES treatment suppressed RIP140 expression and down-regulated NF-κB activity, which suggested that ES exhibited anti-inflammatory and anti-apoptotic effects possibly through inhibition of the RIP140/NF-κB pathway.

In conclusion, the present study demonstrated that ES (20, 40 mg kg⁻¹) administration reduced infarct size and improved cardiac function after I/R injury in rats. The cardioprotective effect of ES may be attributed to its ability to suppress myocardial apoptosis and inflammation, which possibly is at least partially due to the inhibition of the RIP140/NF-κB pathway. Therefore, our findings indicated that ES could be a potential therapeutic or preventive agent for the treatment of cardiovascular diseases.

Acknowledgements

The study was supported by the Foundation for Science of Chinese Medicine, the Foundation for Science of Integrated Chinese and Western Medicine, the National Natural Science Foundation of China (81403041, 81603258), the Natural Science Foundation of Jiangsu Province (BK20140961, BK2016043130), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Footnote

† Tao Weiwei and Zuo Ting equally to this work.

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