Pretreatment with Panaxatriol Saponin Attenuates Mitochondrial Apoptosis and Oxidative Stress to Facilitate Treatment of Myocardial Ischemia-Reperfusion Injury via the Regulation of Keap1/Nrf2 Activity

Myocardial ischemia-reperfusion injury (MIRI) is a type of severe injury to the ischemic myocardium that can occur following recovery of blood flow, and for which, there is no effective treatment. Panaxatriol saponin (PTS), a major active component of P. notoginseng, has been used clinically to treat ischemia-related encephalopathy due to its antioxidant activity, but its effect on ischemic cardiomyopathy and underlying mechanism of action is still unclear. This study was performed to investigate the protective effect of PTS against MIRI and explore the potential underlying mechanisms. Hydrogen peroxide (H2O2) was used to stimulate cardiomyocytes, to mimic MIRI in vitro. Cell viability was tested using the CCK-8 method. The antioxidant activity of PTS in the H9c2 rat cardiomyocyte cell line was examined using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). The levels of superoxide dismutase-1 (SOD1), SOD2, and heme oxygenase (HO-1) were determined by Western blotting and/or immunofluorescence. The antiapoptotic effect of PTS was determined. In addition, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (ΔΨm) changes were assessed. Changes in Keap1/Nrf2 activation were evaluated by Western blotting analysis, molecular docking, and immunoprecipitation. An in vivo MIRI model was established in rats, and the myocardial infarct size was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Myocardial enzyme activities were determined by ELISA or biochemical analyses. Furthermore, changes in Nrf2 activation were evaluated, and the regulatory effect of PTS on cardiomyocyte apoptosis was examined using the Nrf2 blocker, ML385. The results showed that PTS ameliorated the cardiomyocyte injury induced by H2O2, characterized by increased cell viability, decreased reactive oxygen species (ROS) production, and promotion of SOD1, SOD2, and HO1 expression. PTS inhibited cardiomyocyte apoptosis in vivo and in vitro. PTS also reduced mPTP opening and stabilized ΔΨm in H9c2 cells. Molecular docking and immunoprecipitation study revealed that PTS can disrupt Keap1/Nrf2 interaction by directly blocking the binding site of Nrf2 in the Keap1 protein. In vivo, PTS decreased the area of myocardial infarction and attenuated pathological damage in ischemia-reperfusion (I/R) rats. In addition, the activities of myocardial injury markers were decreased by PTS. Finally, PTS regulated nuclear translocation of Nrf2, and ML385 blocked the therapeutic effect of PTS in vivo and in vitro. These results suggested that PTS has therapeutic potential for MIRI by targeting Keap1/Nrf2 activity.


Introduction
Coronary heart disease (CHD) is the leading cause of death and disability worldwide [1] and is usually attributable to the detrimental effects of acute myocardial ischemia-reperfusion injury (MIRI) [2]. MIRI usually occurs in patients with acute ST-segment elevation myocardial infarction (STEMI). In these patients, the most effective therapeutic intervention to reduce acute myocardial ischemic injury and limit the scale of myocardial infarction is timely and effective myocardial reperfusion via primary percutaneous coronary intervention (PPCI) or thrombolytic therapy [2]. However, myocardial reperfusion itself induces further death of myocardial cells.
Several mechanisms have been proposed to contribute to reperfusion injury. It is generally accepted that oxidative stress is a major contributor to the onset and development of MIRI [3,4]. Oxidative stress triggered by excessive reactive oxygen species (ROS) is considered an essential initiator of cardiomyocyte apoptosis. Therefore, antiapoptotic treatments represent a promising research direction for the development of therapeutic strategies for ischemic cardiomyopathy; it is possible to control the disease process and protect the functional reserve of the myocardium [5][6][7].
Mitochondrial dysfunction has been reported to be closely related to ischemic heart disease [8][9][10]. Under conditions of myocardial ischemia and hypoxia, mitochondria mainly achieve energy metabolism through anaerobic glycolysis, which leads to the production of large amounts of lactic acid; this causes intracellular acidosis, which in turn damages mitochondria. During myocardial reperfusion, due to the instantaneous increase in local blood oxygen concentration, ROS bursts occur in mitochondria and further undermine the dynamic balance between oxidation and reduction, leading to mitochondrial permeability transition pore (mPTP) opening, abnormal distribution of charged ions inside and outside the membranes, and disruption of electrochemical gradients, thus causing mitochondrial membrane potential (ΔΨm) loss.
Numerous natural plants have been reported to exhibit antioxidant activity, and recent studies have indicated their therapeutic potential for ischemic cardiomyopathy, including MIRI [11][12][13][14]. Panax notoginseng, the root of Panax notoginseng (Burk.) F.H. Chen, has been used as a traditional herbal medicine in China for more than 600 years due to its beneficial effects in preventing and treating various diseases, including cardiovascular and cerebrovascular diseases [15]. Panaxatriol saponin (PTS), one of the major effective components of P. notoginseng, has been used clinically in China for the treatment of cerebral diseases due to its antioxidant, anti-inflammatory, antiplatelet, and angiogenesis-promoting activities [16][17][18][19]. Furthermore, an early study indicated that pretreatment with ginseng total saponin ameliorated ischemia-reperfusion (I/R)-induced myocardial damage, and this protective effect was mediated by a decrease in oxidative stress [20]. However, the specific effects of PTS on MIRI, and the underlying mechanism of action, remain largely unknown.
The present study was performed to investigate the protective effect of PTS on MIRI, and to identify the potential mechanisms underlying these effects. The results indicated that PTS pretreatment attenuated oxidative stress-induced cardiomyocyte apoptosis and protected the heart against MIRI. The cardioprotective effects of PTS involved reinforcement of the antioxidant system via the activation of Nrf2.
1.1. Reagents. PTS extracted from P. notoginseng was obtained from Huasun Group Co., Ltd. (Sichuan, China). The main components of PTS were the ginsenoside Rg1, notoginsenoside R1, and ginsenoside Re (Supplementary Materials: Figure S1). The animal experiments had two stages. In the first stage, a total of 60 rats were divided into the following six groups (n = 10 per group) to determine the optimal concentration of PTS: I/R group, 60 min of ischemia followed by 24 h of reperfusion; sham group, the same operation as the I/R group, but the left anterior descending (LAD) branch was not ligated; sham+PTS group, intragastric administration of 100 mg/kg/d PTS aqueous solution and the same operation as in the sham group; I/R+PTS groups, intragastric administration of 25, 50, or 100 mg/kg/d PTS aqueous solution followed by I/R operation. The pretreatment was carried over 7 days. The sham and I/R groups were pretreated with vehicle (water) before the operation. In the second stage, a total of 50 rats were divided into five groups (n = 10 per group) to explore the underlying mechanisms: I/R group, same as the first stage; sham group, same as the first stage; sham+PTS group, same as the first stage; I/R +PTS group, treatment with the optimal concentration of PTS determined in the first stage, and I/R as in the first stage; PTS+ML385 group, treatment with the optimal concentration of PTS 30 min after intraperitoneal injection of ML385 at a dose of 30 mg/kg [21].

Establishment of the Rat Myocardial I/R Injury Model.
The rat myocardial I/R injury model was produced as described previously [22]. Briefly, rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (50 mg/kg). The left ascending artery was occluded using a 7-0 silk suture tied transiently over PE-10 tubing for 60 min, after which the knot on the PE-10 tubing was cut. Successful ischemia was determined by the elevation of the ST segment on electrocardiography (ECG). Sham-operated control animals underwent the same surgical procedures, with the exception of LAD coronary artery occlusion.    The absorbance of the sample at 450 nm was measured using a microplate reader.
1.9. Isolation and Culture of Neonatal Rat Primary Cardiomyocytes. Neonatal rat primary cardiomyocytes (NPCMs) were obtained from Sprague-Dawley rats at 48 ± 6 h after birth, as described previously [24]. Briefly, rats were euthanized by decapitation, and the heart was removed     Oxidative Medicine and Cellular Longevity and placed in a culture dish containing Hank's balanced salt solution (HBSS) on ice. The hearts were digested with 1% type II collagenase and trypsin (2 : 1) without EDTA. After 1 h of natural sedimentation, the myocardial fibrocytes were discarded to obtain ventricular myocytes. The cells began to beat synchronously 1-2 days after plating, and the experiment was initiated. Cells were maintained in DMEM with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/ mL streptomycin, and cultured at 37°C in an atmosphere of 5% CO 2 .
1.10. TUNEL Assay. Apoptosis was assessed by TUNEL assay in accordance with the manufacturer's instructions (Yeasen, Shanghai, China). Briefly, samples were fixed in 4% paraformaldehyde for 20 min, and then incubated in 0.1% Triton X-100 for 30 min, and covered with TUNEL reaction mixture. The samples were then incubated in a humidified chamber for 1 h at 37°C in the dark and then subjected to TUNEL staining. Finally, apoptotic cells were visualized using an inverted fluorescence microscope and counted in four randomly selected fields in each group.

Transmission Electron Microscopy.
Transmission electron microscopy (TEM) was performed as described previously [25]. Briefly, cardiac tissue was dissected into 1 mm 3 piece and fixed in 4% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.2) overnight at 4°C. Following several washes in buffer, the samples were post-fixed with 2% osmium tetroxide and 1% uranyl acetate for 2 h, rinsed in water, dehydrated in an ascending ethanol series followed by 100% acetone, and then infiltrated and embedded in Eponate. Ultrathin sections were cut on a microtome and mounted onto 200-hex-mesh copper grids. The sections were exposed to the primary stain (5% aqueous uranyl acetate) followed by the secondary stain (lead citrate) and then visualized by TEM (H-600IV; Hitachi, Tokyo, Japan).      (Figures 1(d) and 1(e)). In addition, the activity of the antioxidant enzyme, superoxide dismutase-1 (SOD1), was significantly decreased after exposure to H 2 O 2 (Figures 1(f) and 1(g)). Next, the antioxidant activities of PTS in H9c2 cells were examined. We first analyzed the effect of PTS on cell viability in the presence of H 2 O 2 (200 μM) and found that the reduction in cell viability was markedly ameliorated by pretreatment with PTS for 12 h at a concentration of 10 μg/mL (Figure 1(h)). Therefore, this PTS concentration was chosen for subsequent experiments.
To confirm that the effects of PTS on H9c2 cell survival were caused by ROS inhibition, the abovementioned experiments were performed in the presence of 10 μg/mL PTS. H 2 O 2 significantly increased the ROS components of the cells and impaired their antioxidant capacity, which was reflected in increased DCFH-DA fluorescence intensity and decreased SOD1 fluorescence intensity and protein expression. However, PTS pretreatment significantly ameliorated the dysregulation of antioxidant capacity of H9c2 cells, including downregulation of ROS (Figures 1(i) and 1(j)) but upregulation of SOD1, SOD2, and HO1 which are the major components of antioxidant enzyme (Figures 1(k)-1(q)). Taken together, these observations confirmed the ability of H 2 O 2 to induce oxidative stress damage in cardiomyocytes and the therapeutic effect of PTS.    (Figures 2(a) and 2(b)). Similar results were obtained by TUNEL assay using an inverted fluorescence microscope, indicating that PTS ameliorated the marked elevation of TUNEL fluorescence intensity by H 2 O 2 stimulation (Figures 2(c) and 2(d)). Moreover, the levels of apoptosis-related proteins, including cleaved caspase-3,

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Oxidative Medicine and Cellular Longevity cleaved PARP-1, Bax, and cytochrome-c (Cyt-c), were analyzed. Our data demonstrated that the levels of cleaved caspase-3, Bax, and cleaved PARP-1, as well as Cyt-c, were significantly higher in the H 2 O 2 than vehicle and PTS alone groups at 12 h after treatment. However, the changes in these indicators compared with the H 2 O 2 group were markedly ameliorated in the PTS+H 2 O 2 group (Figures 2(e) and 2(f )) (Supplementary materials: Figure S2). Furthermore, similar results were obtained in NRCMs (Figures 2(g) and 2(h)). The above results suggested that PTS ameliorated H 2 O 2 -induced cardiomyocyte apoptosis.

Effects of PTS on mPTP Opening and Mitochondrial
Membrane Potential (ΔΨm) of H9c2 Cells. Mitochondrial dysfunction is one of the main pathways of apoptosis and thus contributes to I/R injury. In the process of apoptosis, the permeability of the inner mitochondrial membrane increases, and the influx of Ca 2+ leads to loss of the mitochondrial transmembrane potential. This change is mainly caused by the opening of mPTPs between the inner and outer mitochondrial membranes. Therefore, we investigated mPTP opening in H 2 O 2 -treated H9c2 cells by monitoring the fluorescence of mitochondrial-entrapped calcein using the calcein AM and CoCl 2 coloading method [28]. As shown in Figures 3(a) and 3(b), the fluorescence intensity of mitochondrial calcein was significantly decreased in the H 2 O 2 group compared with the vehicle group, indicating that mPTP opening was enhanced following H 2 O 2 treatment. Surprisingly, pretreatment with PTS markedly increased the fluorescence intensity compared with the H 2 O 2 group, suggesting that PTS inhibited H 2 O 2 -induced mPTP opening in H9c2 cells. Next, we used MitoTracker Red CMXRos and Hoechst costaining to directly observe mitochondrial activity, to determine the changes in ΔΨm in H9c2 cells. Similarly, the fluorescence intensity of MitoTracker Red CMXRos was significantly inhibited in apoptotic cells in the H 2 O 2 group, indicating that the ΔΨm level increased following H 2 O 2 treatment. In the H 2 O 2 +PTS group, however, fewer apoptotic H9c2 cells were observed, as indicated by bright Hoechst staining. PTS pretreatment also markedly increased the fluorescence intensity compared with the H 2 O 2 group (Figures 3(c)

PTS Binds to the Kelch Domain of Keap1 to Regulate
Nrf2 Activity. The Keap1/Nrf2 signaling is regarded as one critical endogenous antioxidative stress pathway discovered so far. Previous studies indicated that Nrf2 translocates from the cytoplasm to the nucleus, where it regulates the expression of antioxidant genes under conditions of stress [29]. To gain mechanistic insights into PTS-induced inhibition of oxidative stress in vitro, we next investigated the Keap1/ Nrf2 signaling in H9c2 cells. We first analyzed Nrf2 levels in the cytoplasm and nucleus of H 2 O 2 -treated H9c2 cells. The results indicated that cytoplasmic Nrf2 level increased, and the nuclear Nrf2 level decreased, after H 2 O 2 treatment. Conversely, PTS promoted Nrf2 nuclear translocation from the cytoplasm to the nucleus (Figures 4(a)-4(c)). Since these data revealed the antioxidant stress activity of PTS is probably associated with the regulation of the Nrf2 activity. And it has been well understood that Kelch-like ECH-associated protein-1 (Keap1) ubiquitinates Nrf2 to promote its  Figure 6: PTS attenuates myocardial ischemia/reperfusion injury (MIRI) in rats. A total of 60 rats were divided into six groups to determine the optimal concentration of PTS to treat MIRI rats, including the I/R group, sham group, sham+PTS group, and I/R+PTS groups (intragastric administration of 25, 50, or 100 mg/kg/d PTS aqueous solution followed by I/R operation). The pretreatment was carried over 7 days. The sham and I/R groups were pretreated with vehicle (water) before the operation.    (Figure 6(a)). As shown in Figures 6(b) and 6(c), I/R rats showed an increase in myocardial INF compared with the sham group. However, PTS pretreatment significantly decreased INF in comparison with the I/R group. In addition, the I/R group showed obvious myocardial fiber fracture, cellular edema, hemorrhage, necrosis, and neutrophil infiltration. Interestingly, compared with the I/R group, myocardial tissue damage showed gradual recovery with increasing doses of PTS (25, 50, and 100 mg/kg) (Figure 6(d)). I/R significantly induced myocardial injury in rats, as evidenced by the increased serum levels of MB, cTn-T, CK, LDH, and CK-MB. Surprisingly, these abnormal markers were markedly attenuated by pretreatment with PTS (100 mg/kg) (Figures 6(e)-6(i)). Taken

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Oxidative Medicine and Cellular Longevity together, these observations support the potential value of PTS for the treatment of MIRI.

PTS Ameliorated Myocardial Mitochondrial Apoptosis in
MIRI Rats by Regulating Nrf2. Next, we examined the potential mechanisms underlying the cardioprotective effects of PTS. As the data presented above indicated the antiapoptotic effect of PTS associated with regulation of Nrf2 nuclear translocation, we blocked the activity of Nrf2 and performed I/R surgery according to the same procedure. First, sections of paraffin-embedded rat heart tissue were subjected to TUNEL staining, and the results indicated that I/R markedly induced myocardial apoptosis in rats, as evidenced by the increased TUNEL fluorescence intensity. PTS pretreatment alleviated the cardiomyocyte apoptosis caused by I/R, and this effect was diminished by ML385 administration (5 μM) (Figures 7(a) and 7(b)). Next, the levels of apoptosis-related proteins were analyzed. Western blotting analysis showed that the levels of cleaved caspase-3 and cleaved PARP-1, as well as Cyt-c, were significantly higher, while the level of Bax was significantly lower, in the I/R group compared with the sham and sham+PTS groups. However, the I/R+PTS+ML385 group showed no significant differences in the expression level of these proteins in comparison with the I/R group, indicating that the therapeutic effect of PTS was attenuated by inhibition of Nrf2 (Figures 7(c)-7(g)). Furthermore, representative TEM images of cardiac mitochondria showed rupture of the outer mitochondrial membranes, disappearance of cristae, and even vacuolization in the I/R group. Interestingly, more complete outer mitochondrial membrane structures and cristae, and less vacuolization, were observed in the I/R +PTS group compared with the I/R group, but these differences were inhibited by ML385 administration (Figure 7(h)). Taken together, the above results confirmed that PTS can rescue I/R rat cardiomyocyte mitochondrial apoptosis by regulating Nrf2. Oxidative Medicine and Cellular Longevity