Tubeimoside I Ameliorates Myocardial Ischemia-Reperfusion Injury through SIRT3-Dependent Regulation of Oxidative Stress and Apoptosis

Myocardial ischemia-reperfusion injury (MIRI) is a phenomenon that reperfusion leads to irreversible damage to the myocardium and increases mortality in acute myocardial infarction (AMI) patients. There is no effective drug to treat MIRI. Tubeimoside I (TBM) is a triterpenoid saponin purified from Chinese traditional medicine tubeimu. In this study, 4 mg/kg TBM was given to mice intraperitoneally at 15 min after ischemia. And TBM treatment improved postischemic cardiac function, decreased infarct size, diminished lactate dehydrogenase release, ameliorated oxidative stress, and reduced apoptotic index. Notably, ischemia-reperfusion induced a significant decrease in cardiac SIRT3 expression and activity, while TBM treatment upregulated SIRT3's expression and activity. However, the cardioprotective effects of TBM were largely abolished by a SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP). This suggests that SIRT3 plays an essential role in TBM's cardioprotective effects. In vitro, TBM also protected H9c2 cells against simulated ischemia/reperfusion (SIR) injury by attenuating oxidative stress and apoptosis, and siSIRT3 diminished its protective effects. Taken together, our results demonstrate for the first time that TBM protects against MIRI through SIRT3-dependent regulation of oxidative stress and apoptosis. TBM might be a potential drug candidate for MIRI treatment.


Introduction
Acute myocardial infarction (AMI) induced by coronary artery occlusion remains a leading cause of morbidity and mortality worldwide [1]. And it brings serious psychological and economic burden to patients who suffer from the condition. Early reperfusion therapy, such as thrombolysis and percutaneous coronary intervention (PCI), has become a routine treatment strategy for myocardial infarction [2]. However, reperfusion may lead to irreversible damage to the myocardium and increases mortality in AMI patients. This phenomenon is called myocardial ischemiareperfusion injury (MIRI) [3]. The underlying mechanisms of MIRI are complex. The main involved mechanisms include overproduction of reactive oxygen species (ROS), calcium overload, inflammatory reactions, mitochondrial dysfunction, and activation of apoptosis [4][5][6][7][8]. Moreover, more and more evidences show that ROS is the major pathological process leading to MIRI [4,9]. ROS are produced mainly by mitochondria, and excessive ROS leads to apoptosis of cardiomyocytes [8,10]. Therefore, it is a vital preventional treatment of MIRI to reduce the apoptosis induced by oxidative stress during reperfusion.
In the present study, we observed that TBM alleviated IR-induced oxidative stress and apoptosis both in vivo and in vitro. And its protective effects were abolished by SIRT3 inhibition. These results demonstrated that TBM ameliorated MIRI through SIRT3-dependent regulation of oxidative stress and apoptosis, indicating TBM is a promising drug to treat MIRI.

Animal Experiments.
Wild-type male C57BL/6 mice were used in this study. The laboratory animal center of Chongqing Medical University supplied mice. Mice were kept under standard specific pathogen-free conditions and were allowed free access to water and chow. Animal experiments were approved by the Animal Ethic Committee of Chongqing Medical University. Mice were randomly divided. The following groups were set up: (1) sham group: mice underwent the sham operation (all operations were the same with those in the IR group except ligation) and were treated with vehicle (5% saline); (2) TBM group: TBM (4 mg/kg) was given via intraperitoneal injection; (3) IR group: mice underwent IR operation and treated with vehicle (5% saline); (4) IR+TBM group: mice underwent IR operation and treated with 4 mg/kg TBM via intraperitoneal injection at 15 min after IR operation; and (5) IR+TBM+3-TYP group: mice were pretreated with 3-TYP (a dose of 50 mg/kg every 2 days for a total of three doses was intraperitoneally injected prior to the IR surgery), treated with TBM (4 mg/kg via intraperitoneal injection at 15 min after IR operation) and then surgery was performed as previously described [32]. Briefly, mice were anesthetized with 2-3% isoflurane. Animals were intubated orally and connected to a mouse miniventilator (MiniVent 845, Harvard Apparatus, Holliston, MA, USA) with PE-90 tubing. To keep the mice under anesthesia, 1% isoflurane was continuously provided.

Oxidative Medicine and Cellular Longevity
Core body temperature was maintained at 37°C using a thermo heating pad and monitored with a rectal thermometer. A median sternotomy was performed, and the left anterior descending coronary artery (LAD) was visualized. An 8.0 nylon suture (Prolene; Ethicon, Norderstedt, Germany) was placed around the vessel and a loose loop was formed to induce ischemia. For easier ligature release and better reperfusion, a PE-10 tube was placed on the LAD before loop was tied. Ligation was visually confirmed by appearance of pale and bulging myocardium in the area at risk. The PE-10 tube was removed after 30 minutes to allow reperfusion of the myocardium which was indicated by a bright red color  Figure 3: Effect of TBM on cardiac apoptosis and apoptotic signaling pathway in IR. TBM (4 mg/kg) was given at 15 min after ischemia via intraperitoneal injection. After reperfusion, cardiac tissues were detected using TUNEL assay. Total proteins, cytoplasmic proteins, and mitochondrial proteins were extracted and expression of Cyt-Cyto C, Cyt-GAPDH, Bax, Bcl-2, Cleaved Caspase-3, GAPDH, Mito-Bax, and COX-IV was measured using western blotting. (a) Representative images of TUNEL assay. Apoptotic cardiomyocytes were labeled using TUNEL staining, and DAPI was used to detect nuclei. Scar bar: 150 μm. All numerical data are expressed as means ± SD, for apoptotic index n = 6; for western blotting, n = 4. * p < 0:05; * * p < 0:01. 5 Oxidative Medicine and Cellular Longevity within the LAD. Three hours after reperfusion, supernatant plasma and the hearts were harvested and stored at −80°C for subsequent tests. For cardiac function, apoptotic index, and infarct size determination, all sutures and tubes were taken out and incisions were sutured after ischemia, and then, mice were set free and reperfusion lasted for 24 h. Details of in vivo experimental protocol are shown in Figure S1.
2.3. Cell Culture and Treatment. H9c2 cells were cultivated as described previously [33]. Simulated ischemiareperfusion (SIR) was performed using ischemic DMEM (DMEM without glucose and serum and dissolved oxygen in the culture media was expelled by filling nitrogen) and low-oxygen incubator as described previously [33][34][35][36][37]. Briefly, H9c2 cells were inoculated and cultivated for 24 h, starved for 4 h, and pretreated with TBM for 1 h. Then, cells      7 Oxidative Medicine and Cellular Longevity were treated in ischemic DMEM and incubated in lowoxygen incubator (Thermo Scientific, Waltham, MA, USA) for 2 h (air conditions are 5% CO 2 , 1% O 2 , and 94% N 2 ) and reperfusion was initiated by changing ischemic DMEM into complete DMEM (containing 4.5 g glucose and 10% FBS) and incubated using 95% air+5% CO 2 . Cells were harvested at 4 h (for oxidative stress measurement and western blotting) or 24 h (for cell viability and apoptotic index detection) after reperfusion. Figure S2 shows the detailed in vitro experimental protocol.

Echocardiography.
Echocardiography was conducted to assess the cardiac function at 24 h after reperfusion as described before [33]. Briefly, mice were anaesthetized and laid on handling platform warmed to 37°C. Hair removing cream was used to remove the chest fur. Ultrasound probe (L8-18i-D PROBE, GE Healthcare, Boston, MA, USA) was used to image heart long axis. Then, EDV (end diastolic volume), ESV (end systolic volume), LVIDs (left ventricle internal diameter at systole), and LVIDd (left ventricle internal diameter at diastole) were measured with M-mode images. The LVEF (left ventricle ejection fraction) and LVFS (left ventricle fractional shortening) were calculated according to instrument instructions. An investigator blinded to the treatment performed these measurements.

TTC Staining.
Myocardial infarct size was measured by TTC staining. Briefly, after 30 min of ischemia and 24 h of reperfusion, mouse hearts were excised and frozen at -20°C for 15 min. Then, hearts were cut into 1 mm thick transverse slices, which were incubated in 2% TTC (Solarbio, Beijing, China) at 37°C for 10 min. Images were photographed using a digital camera. The areas of infarcted myocardium (white region) and viable tissue (red region) were evaluated using Image Pro Plus (Media Cybernetics). The degree of infarct was expressed as a percentage of infarcted myocardium/the LV area.
2.6. MDA, SOD, and LDH Measurements. To determine systemic oxidative stress, MDA, SOD, and LDH measurements were carried using commercial kits. The Multiskan Spectrum Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to read spectrophotometrical values. MDA, SOD, and LDH concentrations were calculated following manufacturer's instructions.

Western
Blotting. Samples were lysed in ice-cold lysis buffer for 30 min and centrifugated at 12,000 × g for 15 min. For mitochondrial protein extraction, commercial kits were used. Mitochondria/Cytosol Fractionation Kits (C3601 for cells, C3606 for tissues) were bought from Beyotime (Shanghai, China), and mitochondrial or cytoplasmic proteins were extracted according to the manufacturer's instruction. Protein concentration was determined using the Bradford method. Finally, target proteins were determined with western blotting. Detailed western blotting protocol can be referred from previous literatures [33,38,39].

Terminal Deoxynucleotidyl Transferase dUTP Nick End
Labeling (TUNEL) Assay. TUNEL assay was used to determine apoptotic cardiomyocytes.
Briefly, 4% paraformaldehyde-fixed myocardial tissues were embedded with paraffin and sectioned into 5 μm thick sections. Sections were put onto slides and deparaffinized with xylene. To increase permeability, sections were treated with proteinase K solution (20 μg/ml, 37°C, 30 min) and 0.5% Triton X-100 (room temperature, 5 min). Then, DNA breaks were labeled in TUNEL reaction mixture (37°C, 60 min, lightfree). After being rinsed with PBS for 3 times, slides were washed with PBS and the nuclei were labeled with DAPI. Fluorescence microscopy was used to image sections.
2.12. Statistical Analysis. All numerical data are expressed as means ± standard deviation (SD). GraphPad Prism 8.0 software was used to perform all analyses. Student's t-test was used to evaluate differences between two groups, and oneway analysis of variance (ANOVA) followed by Dunnett's post hoc test was used to evaluate differences among multiple groups. p < 0:05 was considered as statistically significant.

TBM Improves Cardiac Function and Reduces Infarct
Size and Plasma LDH Level in IR Mice. As Figures 1(a)-1(c) show, IR induced a significant reduction in LVEF and LVFS compared with the sham group, while TBM improved cardiac function by increasing LVEF and LVFS. To detect TBM's effects on the heart more directly, TTC staining was used to detect myocardial infarction size and plasma LDH levels were also measured. As shown in Figures 1(d)-1(f), infarct size and plasma LDH levels increased by IR were significantly reduced by TBM.

TBM Reduces IR-Caused Cardiac Oxidative Stress In
Vivo. As Figure 2(a) shows, MDA content in cardiac tissue was increased by IR, indicating that IR induced oxidative stress in the heart. IR-induced oxidative stress was significantly inhibited by TBM (Figure 2(a)). Generally, oxidative stress is caused by the imbalance between ROS production and scavenging ability. To figure out TBM's effects on oxidative stress, we detected TBM's effects on both ROS production and ROS scavengers. On the one hand, TBM decreased ROS production by inhibiting NOX2 which is an essential superoxide producer and contributes to oxidative stress under various pathological conditions (Figures 2(c) and 2(f)). On the other hand, TBM increased SOD activity (Figure 2(b)) and enhanced expression of antioxidant factors Nrf2 and NQO1 (Figures 2(c), 2(g), and 2(h)). As SIRT3 can elevate activity of SOD2 by deacetylation, we also detected SIRT3 and Ac-SOD2 by western blotting. As Figures 2(c)-2(e) showed, IR-inhibited SIRT3 and IR-enhanced Ac-SOD2/SOD2 were all reversed by TBM treatment. These results indicated TBM reduced IR-caused cardiac oxidative stress in vivo.

TBM Reduces IR-Caused Cardiac Apoptosis and Inhibits
IR-Activated Apoptotic Signaling Pathway. Reperfusion induces a burst of ROS, and ROS acts on mitochondria, destroys integrity of mitochondria, and releases cytochrome C into the cytoplasm. Cytoplasmic cytochrome C (Cyt-Cyto C) causes apoptosis of cardiac myocytes. As shown in Figure 3, IR induced apoptosis and increased expression of apoptotic proteins, while all these alterations were significantly reversed by TBM. As SIRT3 can inhibit apoptosis by preventing translocation of Bax and TBM induced expression of SIRT3, we supposed that TBM could prevent translocation of Bax. Indeed, IR-increased mitochondrial-located Bax level was significantly inhibited by TBM (Figures 3(c) and 3(g)). These results indicated that TBM reduced IRcaused cardiac apoptosis in vivo.
Then, the influence of 3-TYP on TBM's effects in IR mice was examined. As Figure 4 showed, 3-TYP attenuated TBM's cardioprotective effects, because TBM-increased LVEF and LVFS were attenuated by 3-TYP. Also, compared with the IR+TBM group, mice in the IR+TBM+3-TYP group had increased infarct size and LDH levels. These 9 Oxidative Medicine and Cellular Longevity results indicated that TBM protected the heart through SIRT3.

TBM's Effects on SIR-Injured H9c2
Cells' Cell Viability, Oxidative Stress, and Apoptosis. To illuminate the underlying molecular mechanism, we performed in vitro studies using H9c2. First, TBM's effects on normal H9c2 cells' cell viability were examined. As shown in Figure 7(a), TBM had no significant effect on viability of normal H9c2 cells. Then, we treated SIR-injured H9c2 cells with TBM at different concentrations and found that TBM increased cell viability significantly (Figure 7(b)). As 4 μM TBM promoted cell viability most effectively, a dose of 4 μM TBM was used in further mechanism investigations.  (Figure 7(d)). Also, detection of ROS using fluorescent staining demonstrated that TBM could reduce ROS in SIRinjured H9c2 cells (Figures 7(e) and 7(f)). Further, we detected expression of proteins related with redox balance. As shown in Figures 8(a)-8(f), SIR-decreased expression of SIRT3, Nrf2, and NQO1 and increased expression of oxidative protein NOX2 brought by SIR were reversed by TBM.

SIRT3 Is an Essential Factor in TBM's Protective Effects in SIR-Injured H9c2
Cells. To figure out SIRT3's role in TBM's protective effects in SIR-injured H9c2 cells, SIRT3 was knocked down by siRNA. First, three siRNAs were designed and synthesized, and their efficiency and specificity were confirmed by western blotting. As Figure 9(a) showed, SIRT3 could be knocked down by all three siRNAs efficiently and specifically. Then, the role of SIRT3 in TBM's protective effects was examined. As Figures 9(b)-9(h) showed, TBM's prosurvival, antioxidative, and antiapoptotic effects were all reduced by siSIRT3. Additionally, siSIRT3 abolished TBM's inhibitive effects on NOX2 expression and Ac-SOD2/SOD2 ratio (Figures 10(a)-10(c)). Also, siSIRT3 attenuated enhanced expression of antioxidative proteins Nrf2 and NQO1 brought by TBM (Figures 10(e) and 10(f)).

Discussion
In this study, we examined TBM's effects on MIRI both in vivo and in vitro. In vivo, TBM administration improved postischemic cardiac function, decreased myocardial infarct size, reduced plasma LDH levels, reduced oxidative stress, and reduced apoptotic cardiomyocyte number. In vitro, TBM improved cell viability, decreased oxidative stress, and reduced apoptotic H9c2 number. TBM's protective effects are mediated, at least partially, by activating SIRT3, as both 3-TYP, a specific SIRT3 inhibitor, and siSIRT3 abolished TBM's protective effects remarkably.
It is well accepted that oxidative stress plays a central role in MIRI. Increased ROS production and decreased ROS scavenging ability lead to oxidative stress [40]. In this study, TBM protected against MIRI by reducing oxidative stress. On the one hand, TBM decreased ROS production by inhibiting NOX2. On the other hand, TBM increased antioxidant factors such as Nrf2 and NQO1. As far as we know, this is the first report that TBM is of antioxidative activity.
Among 7 sirtuins, SIRT3 has gained more attention due to its mitochondrial location and involvement in extending human lifespan [41]. In MIR rats, SIRT3 is downregulated [17]. Upregulation of SIRT3 ameliorates MIRI through inhibiting mPTP opening by deacetylating cyclophilin D [21]. Also, SIRT3 deficiency exacerbates MIRI [18]. All these studies demonstrated that SIRT3 played vital role in MIRI. SIRT3 induces Nrf2 which is responsible for transcription of important antioxidant factors. These antioxidant factors fight against oxidative stress [42][43][44]. SIRT3 elevates SOD2's activity by deacetylation [45]. SIRT3 also inhibits apoptosis. First, overexpression of SIRT3 increases the ratio of Bcl-2/Bax [46]. Second, SIRT3 prevents translocation of Bax to mitochondria by deacetylating Ku70 [24]. Third, SIRT3 acetyls cyclophilin D and prevents release of mitochondrial cytochrome C into the cytoplasm [21]. In this study, we found that TBM administration ameliorated MIRI by reducing oxidative stress and apoptosis and all these protective effects were abolished by SIRT3 inhibition. All these results suggested that Sirt3-mediated antioxidative and antiapoptotic mechanisms contributed to TBM's protective effects.
Previous studies on TBM focused on its antitumor activity [27]. And most of these reports demonstrated that TBM induced oxidative stress and apoptosis [47,48]. Our results seem to contradict with previous reports. These contradictions can be explained by SIRT3's diversified functions. On the one hand, SIRT3 can inhibit apoptosis. SIRT3 interacts with OGG1, repairs mitochondrial DNA, and protects from apoptotic cell death under oxidative stress [49]. In cardiomyocytes, SIRT3 protects cells from stress-mediated apoptosis by deacetylation of Ku70 [24]. SIRT3 rescues p53induced growth arrest in human bladder tumor-derived EJ-p53 cells [50]. SIRT3 promotes cancer cell survival by inhibiting apoptosis [51,52]. On the other hand, SIRT3 can promote apoptosis. Under basal conditions, SIRT3 promotes apoptosis by regulating cell survival pathways [53]. SIRT3 promotes cancer cell apoptosis through destabilizing HIF1α [54]. SIRT3 promotes hepatocellular carcinoma apoptosis by reducing Mdm2-mediated p53 degradation [55]. In different cell types or under different conditions, SIRT3 can inhibit or promote apoptosis, because that TBM work through SIRT3. It is reasonable that TBM promotes oxidative stress and apoptosis in cancer cells while TBM inhibits oxidative stress and apoptosis in cardiomyocytes. Besides TBM, trans sodium crocetinate, total salvianolic acid, dihydromyricetin, 3-bromo-4,5-dihydroxybenzaldehyde, exendin-4, and melatonin have been reported to protect against MIRI via SIRT3 [56][57][58][59][60][61][62]. All these drugs induce expression of SIRT3, but their mechanisms are different. Total salvianolic acid, exendin-4, and melatonin induce expression of both SIRT1 and SIRT3. In our study, we found that IR decreased expression of SIRT1, but TBM could not reverse expression of SIRT1 ( Figure S5). Recently, TBM has been reported to antagonize Yoda1-evoked Piezo1 channel activation [63]. Also, Piezo1 is proposed to be a vital factor that leads to cerebral ischemia-reperfusion injury [64]. We speculated that TBM induced expression of SIRT3 by binding to Piezo1. This hypothesis will be tested in the future.
With the development of stent interventional therapy, new antiplatelet agents, and antithrombotic agents, myocardial reperfusion therapy has been greatly optimized. But there is still no effective treatment to prevent MIRI. Cyclosporine A, an inhibitor of cyclophilin D (a protein control-ling mPTP's opening), alleviates MIRI in cardiac IR animal models [65,66]. However, a large multicentric clinical trial investigating cyclosporine A's efficacy reveals no protective effects of the drug on clinical outcomes in MI patients [67]. Metoprolol, a β1 receptor blocker, reduces MIRI. But it requires long transporting times [68]. In the present study, our results demonstrate that TBM alleviates MIRI by reducing oxidative stress and apoptosis in a short term. Previously, we have reported that TBM promoted angiogenesis via the eNOS-VEGF signaling pathway [39]. In the long term, TBM's angiogenic effect might promote AMI patients' rehabilitation. All our results indicate that TBM might be a potent new drug against MIRI.
There are four limits that need to be addressed in our study. First, to confirm SIRT3's function, more intensive methods such as genetic knockout mice should be used. Second, to reproduce physiological state, primary cardiomyocytes should be used in in vitro experiments. Third, we did not figure out TBM's direct target. This will be studied in the future. Fourth, SIRT3 has a long isoform (~44 kDa) All numerical data are expressed as means ± SD, n = 4. * p < 0:05; * * p < 0:01.

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Oxidative Medicine and Cellular Longevity and a short isoform (~28 kDa). In mitochondria, the short isoform is more likely to exhibit deacetylase activity and it is the main one [69]. So, we examined SIRT3's short isoform in this study. However, it has been reported that discrepancy of the two SIRT3 isoforms existed in a cardiac hypertrophy model [70]. We will pay attention to these two isoforms in future studies. In a word, for the first time, our data show that TBM administration inhibits IR-induced cardiac dysfunction, oxidative stress, and apoptosis through SIRT3. Our understanding of TBM's effects has been broadened, and this study provided molecular evidence to develop TBM as a therapeutic intervention of MIRI in AMI patients.

Data Availability
All authors confirm that the data supporting the findings of the study are provided within the manuscript and the supplementary file.

Conflicts of Interest
The authors declare that they have no conflicts of interest.  Figure S1: in vivo experimental protocol. Figure S2: in vitro experimental protocol. Figure S3: TBM and 3-TYP's effects on cardiac function, myocardial infarct size, oxidative stress, and apoptosis in sham group mice. TBM (4 mg/kg) or 3-TYP (50 mg/kg) was intraperitoneally injected into sham operation mice, and cardiac function, myocardial infarct size, oxidative stress, and apoptosis were determined after sham operation.