Cardioprotective Effects of Shaosha-7 on Acute Myocardial Infarction Through Sucrose Metabolism Regulation

Abstract

Objective: This study aimed to identify potential target proteins of the Mongolian drug Shaosha-7 (SS-7) for the amelioration of ischemia-reperfusion injury following acute myocardial infarction and to elucidate its comprehensive mechanism of action in cardio-protection. Methods: The toxicity assay was conducted using zebrafish and H9c2 cardiomyocytes, and the protective effects of SS-7 against I/R injury were studied both in vitro and in vivo. A TMT-based quantitative proteomics analysis was performed to explore the global mechanism of SS-7 on I/R injury rats. The therapeutic mechanism of SS-7 was traced and identified using immunohistochemistry and quantitative RT-PCR. Result: Our results suggested that exposure to SS-7 induced developmental toxicity in zebrafish embryos and cardiomyocyte toxicity. SS-7 could effectively improve cardiac function, reverse myocardial pathological changes in I/R rats and protect cardiomyocytes. In addition, omics analyses revealed 66 differentially expressed proteins (DEPs) associated with SS-7 and I/R and core protein targets were screened through protein-protein interaction (PPI) analyses. Gene Ontology (GO) analysis revealed that proteins mainly functioned the regulation of carbohydrate biosynthetic processes and generation of precursor metabolites and energy. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that DEPs were predominantly associated with starch and sucrose metabolism as well as glycolysis/gluconeogenesis. Immunohistochemistry, ELISA, and RT-PCR assays further support the findings of the omics analysis. We highlighted the importance of glucose metabolism in the treatment of I/R and demonstrated that SS-7 can be used as a novel targeted therapy for I/R. Conclusion: The present study discovered that SS-7 protects rats from I/R injury by influencing glucose metabolism. Our experimental results may provide a novel therapeutic target for I/R injury.

Introduction

With high morbidity and mortality, acute myocardial infarction (AMI) is the most common cardiovascular disease. ¹ In recent years, the treatment of AMI has included arterial bypass grafting and thrombolytic therapy. ² The establishment and popularization of these methods enable the ischemic heart affected by AMI to regain blood perfusion and restore oxygen supply in a short time. ³ Early opening of the occluded coronary artery and rapid recovery of coronary blood flow can reduce the extent of myocardial necrosis, protect and save ischemic myocardium, ⁴ improve prognosis and reduce mortality. ⁵ However, a large number of clinical and laboratory studies have confirmed that reperfusion can further damage the myocardium. ⁶ At present, myocardial injury caused by AMI is recognized as myocardial ischemia/reperfusion (I/R) injury. I/R leads to cardiac dysfunction, such as myocardial stunning, reperfusion arrhythmia, myocyte death, and endothelial and microvascular dysfunction, including the no-reflow phenomenon and inflammatory response, ⁷ which can result in more severe myocardial tissue injury than that caused by the original ischemic insultt.^8,9 At present, although coronary reperfusion can quickly alleviate the symptoms of AMI patients, our understanding of the mechanisms underlying I/R injury is still incomplete, and there is a lack of effective methods to treat myocardial ischemia-reperfusion injury.

Emerging evidence indicates that the energy metabolism has become a potential therapeutic target for AMI. ¹⁰ It has been found that cardiomyocytes predominantly rely on glucose metabolism to produce ATP after acute myocardial infarction. Under ischemia, cardiomyocytes exhibit higher energetic efficiency. ¹¹

Phosphoglycerate kinase (PGK) is a key enzyme in the glycolytic pathway, catalyzing the production of ATP. ¹² PGK1 catalysis the conversion of 1,3-bisphosphoglycerate (1,3-BPG) and ADP to 3-phosphoglycerate (3-BP) and ATP, playing a rate-limiting role in biosynthesis and oxidation. ¹³ Aldo-keto reductase family 1 member B 1 (AKR1B1) is the key rate-limiting enzyme of the polyol pathway, which reduces glucose to sorbitol with the help of reduced coenzyme II (NADPH), and then converts to fructose via sorbitol dehydrogenase (SDH). Numerous studies have demonstrated that AKR1B1 is involved in the development of various cardiovascular disease. Based on these researches, Drugs that effectively inhibit glucose-related molecules such as AKR1B1 or PGK1 can be used to treat I/R.

Mongolian medicine shaosha-7 (SS-7), a well-known Chinese medicinal formula, used to treat cardiovascular disease, is comprised of the herbs Guangzao, clove, aloes, nutmeg, guangmuxiang, ferula and rabbit heart. In our previous study, we found that SS-7 could effectively alleviate I/R injury, and the most effective dosage being 1.6 g/kg. ¹⁴ To date, a cardiac proteomic study investigating the comprehensive mechanism of SS-7's effect on myocardial I/R injury in rats has not been performed.

Systems biology methodologies, including genomics, proteomics and metabolomics, have exhibited significant advantages in studying the specific symptoms related to the comprehensive mechanism of action of herbal formulas. ¹⁵ Using MS relates isobaric chemical labeling of biological samples prior to testing—such as tandem mass tagging (TMT)—is a method of quantitative proteomic analysis. ¹⁶ Using an iTRAQ-based quantitative proteomic approach, 22 proteins were identified in the heart of ischemia rats compared with nonischemic regions. ¹⁷ In addition, 151 ischemia reperfusion related proteins were differentially expressed between that sham operation group and ischemia reperfusion group using a TMT-based quantitative proteomic strategy. ¹⁸ These findings suggest TMT-based quantitative proteomic strategies have the potential to reveal novel diagnostic and therapeutic targets as well as potential biomarkers.

In this present research, we investigated the toxicity of SS-7 at both the zebrafish and cellular levels, and validated its effectiveness at the cellular and animal levels. To explore the comprehensive mechanism of SS-7's effect on I/R rats, we used a TMT based quantitative proteomic method to analyze the specific protein expression levels or protein species in rats’ hearts and verified the relevant molecular expression. We aim to identify potential target proteins to enhance our understanding of the comprehensive mechanism of SS-7.

Methods and Materials

Preparation of Drugs

SS-7 was obtained from the Affiliated Hospital of Inner Mongolia Minzu University and identified by expert pharmacognosists XiangJun Yang. SS-7 was dissolved in a beaker using boiling distilled water, during which the mouth of the beaker was sealed with cling film to minimize contamination. The solution was stirred for 4 h and filtered several times until the drugs was removed, resulting in a mother liquor with a concentration of 0.5 g/mL.

Toxicity Assay of Zebrafish

The reporting of this study conforms to ARRIVE 2.0 guidelines. ¹⁹ Zebrafish (Danio rerio, AB series) were conducted in a recirculating aquaculture system (Beijing Aisheng Company), with each feeding regimen consisting of pellets in the morning and evening and plumpy shrimp. Males and females were maintained under a light-dark cycle at 28.5 ± 1 °C, pH 7.0-7.6, conductivity 440-640 μS, and 14:10 h, respectively. For breeding, healthy, mature zebrafish were transferred to the mating pool in a ratio of 1 female to 2 males. The next morning, zebrafish fertilized embryos were obtained within half an hour after the lights were turned on. Fertilized embryos were washed in deionized water and screened under a somatic microscope. Healthy embryos 4 h after fertilization (4 hpf) were selected for the experiment. After washing the embryos twice using ZR solution, 5 mL of ZR solution was added to each well. SS-7 aqueous extracts of 25, 50, 100, 200, and 400 μg/mL were added to six wells, and the embryos were cultured in a thermostatic incubator at 28 °C until 120 hpf. Zebrafish were observed for mortality, heart rate, and deformities under the microscope at 24, 48, 72, and 120 hpf and photographed, respectively.

Cell Culture and Treatment

Rat H9c2 cardiomyocytes were purchased from ATCC (Manassas, VA). H9c2 cardiomyocytes were cultured in DMEM supplemented with 10% FBS and 100 U/mL penicillin/streptomycin in a humidified incubator. The drug toxicity was detected: H9c2 cardiomyocyte was spread in 96-well plates at a density of 1 × 104 per well. After 24 h of cell attachment, 12.5, 25, 50, 100, 200, 400, 800 µg/mL SS-7 was added for 24 h. MTT was added in 10% volume of the supernatant of the cells at 5 mg/mL, and then the cells were incubated in the incubator at 37 °C for 3-4 h. The MTT crystals were dissolved by adding 150 µL of DMSO per well and shaking and mixing for 10 min, and the OD values were measured at 572 and 630 nm. 150 µL of DMSO was added to each well to dissolve the MTT crystals, shaking and mixing for 10 min, and the OD values were detected at 572 and 630 nm. Drug effectiveness was detected : H9c2 cardiomyocytes were treated within/out 25 µmol/L H₂O₂ for 4 h by pre-treatment with water extract of SS-7 at indicated doses (12.5, 25, 50, 100, 200, 400, 800 µg/mL). Cell viability was analyzed using MTT assay.

ROS Assay

H9c2 cardiomyocytes were seeded in 24-well plates overnight and treated with H₂O₂ for 4 h after by pre-treatment with water extract of SS-7 at indicated dose for 24 h. Cells were rinsed with PBS and incubated with 5 μM CM-H2DCFDA for 30 min to load the fluorescent dye.

Animal Experiments

SD rats (weight 280-300 g) were purchased from the Animal Center of Basic Medical College of Jilin University (SCXKs 2011-0004). The animals’ studies in this research were approved by the Affiliated Hospital of Inner Mongolia Minzu University. The rats were housed in a 12-h light/dark cycle at the temperature of 22 ± 2 °C. All rats were fed a standard diet and had access to water ad libitum. The treatment of rats in this experiment followed the NIH Guide for the Care and Use of Laboratory Animals. All protocols were approved by the Institutional Animal Care and Use Committee of Affiliated Hospital of Inner Mongolia Minzu University. According our previous study, ¹⁴ rats were randomly assigned to 4 groups (n = 8 per group): sham operation (SHAM) rats, ischemia/reperfusion (IR) rats, IR + SS-7 rats (1.6 g/kg/d) (intragastric administration for 15 days) and SS-7 rats (1.6 g/kg/d) (intragastric administration for 15 days). Rats with abnormal ECGs were removed prior to the establishment of the I/R model. The rats were anesthetized with 30 g/L sodium pentobarbital (1.5 ml/kg) and ventilated with air after intubation. The tidal volume was set to 7.5 ml/kg, the frequency was set to 70-80 breaths per minute, with a breathing ratio of 5:4. The left anterior descending coronary artery was ligated approximately 5 mm below the aortic origin. During the experiments, the hearts were ligated for 30 min and re-perfused for 120 min. Echocardiographic Detection of Cardiac Function in Rats. Rat hearts were fixed in situ, embedded in paraffin, and subjected to Hematoxylin and eosin (HE) staining and immunohistochemistry (PGK1,1:1000, proteintech).

Biochemical Indicators

Cell culture supernatants were collected and centrifuged at 12.000 rpm. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA), lactic acid and pyruvate were quantified using ELISA kits from Solarbio science & technology (Bei Jing) Co., Ltd according to manufacturer instructions.

Real-time Quantitative PCR

Total RNA was extracted using the TIANGEN animal tissue and H9c2 cardiomyocytes total RNA extraction kit and converted to cDNA using the TIANGEN Fast King cDNA First Strand Synthesis Kit. Real-time quantitative PCR (RT-qPCR) was analyzed on real-time PCR machine using Ace Q Universal SYBR qPCR Master Mix (Vazyme, China), and the relative expression was calculated using the CT method. The relative mRNA expression level of each target gene was calculated using the 2^−ΔΔCT method. The primers for the gene fragment were designed as follows:

PGK1F: 5′-CGGAGACACCGCCACTTG-3′;

PGK1R: 5′-CCCGATGCAGTAAAGACGAG-3′.

AKR1B1F: 5′-TCTTC CCACCTGGAGGACTT-3′

AKR1B1R: 5′-CTGAC CCCCATAGGACTGGA-3′

GAPDHF: 5′-CAACGACCCCTTCATTGACC-3′;

GAPDHR: 5′-CACCCCATTTGATGTTAGCG-3′

Sample Preparation and TMT Labeling

According to the experimental results described above, rats from different groups were screened to perform further proteomic analysis. The hearts of rats in each group were lysed, and proteins were obtained. Protein content was determined using the BCA Protein Assay Kit. Proteins were reduced with 10 mM DTT for 1 h at 30 °C and then alkylated with 40 mM IAM for 15 min at room temperature. Samples were then diluted 4-fold to 25 mM ammonium bicarbonate and trypsin zed overnight at 37 °C with a 1/50 enzyme/substrate ratio. TMT 16-plex labeling reagents (0.8 mg) were each dissolved in 41 μL of absolute ethanol. Each sample containing 100 μg of peptide in a 25 μL volume of TEAB buffer was combined with 41 μL of its respective 16-plex TMT reagent and incubated for 1 h at room temperature. A pooled sample for normalization was prepared by combining 8 μL of 5% quenching reagent added, and the combined sample incubated for a further 15 min.

LC-MS/MS Analysis, Protein Identification and Quantitative Analysis

LC-MS/MS analysis was analyzed by Q Exactive Mass Spectrometer with an EASY-Spray Ion Source (Thermo Fisher Scientific, USA) with an EASY-nLC 1000 System (Nano HPLC, Thermo Fisher Scientific, USA). The LC-MS/MS raw data were searched against Uniprot_RAT (2020.08.13 download) database. The mass spectrum analysis is completed by Orbitrap fusion mass spectrometry, and the original mass spectrum files generated are processed by the software proteome discoverer 2.4. The following parameters were set: trypsin was enzyme, static modification was carbamidomethyl (C), dynamic modification was M oxidation (15.995 Da) and acetyl (protein N-terminal), species was rat, precursor ion mass tolerance was ± 15 ppm, fragment ion mass tolerance was ± 0.02 Da and max missed cleavages was 2.

Bioinformatics Analysis

Functional annotations of differentially expressed proteins were performed through Gene Ontology (GO) enrichment analysis (http://www.geneontology.org/). In addition, KEGG ²⁰ （http://www.genome.jp/kegg）was used to analyze canonical pathways, and biological networks associated with the lists of differentially expressed proteins.

Statistical Analysis

Data are shown as mean ± SD. SPSS version 17.0 software (IBM) was used for statistical analysis. In this research, a two-tailed Student's t test was used to compare two groups, while a one-way ANOVA with post-hoc testing (Dunnett's multiple comparison) was used to compare multiple groups or each group with a control group. P < .05 was indicates a statistically significant difference.

Result

Acute Toxicity of SS-7 to Zebrafish Larvae

At each SS-7 concentration exposure endpoint, the morphological changes of zebrafish embryos were recorded at 24 hpf and 120 hpf. It was observed that there were no significant changes in heart rate and spontaneous tail coiling frequency of zebrafish embryos within the concentration range of 25 μg/mL to 400 μg/mL (Figure 1A, C). However, discernible lethal effects on zebrafish embryos were found the concentration of SS-7 above 100 μg/mL (Figure B).

Figure 1.

SS-7 Exposure Induces Developmental Toxicity of Zebrafish Embryos. A: The Morphological Development of SS-7 Treatment Groups (25 μg/mL to 400 μg/mL) at 24 hpf and 120 hpf. The Survivorship Curve (B) and Heart Rate (C) of SS-7.

SS-7 Protect H9c2 Cardiomyocytes from H₂O₂ Induced Cardiomyocytes Injury

As shown in Figure 2A, a flow chart of the cellular experiment is display. There is no cardiotoxicity in the concentration range of 12.5 μg/mL to 400 μg/mL of water extract of SS-7 (Figure 2B). Additionally, we found that the water extract of SS-7 effectively suppressed H₂O₂-induced cardiomyocytes injury in H9c2 cardiomyocytes in a concentration-dependent manner (Figure 2C, D). Therefore, the concentration gradients of SS-7 (25 μg/mL, 50 μg/mL, 100 μg/mL) were used in the following experiments.

Figure 2.

SS-7 Inhibited H₂O₂-Induced Cardiomyocytes Injury in H9c2 Cardiomyocytes. A: Schematic Diagram of H9c2 Cardiomyocytes Treated with SS-7 or/and H₂O₂. B: H9c2 Cardiomyocytes were Treated with Different Doses of SS-7. C: H9c2 Cardiomyocytes were Treated at the Indicated Dosed of SS-7 and H₂O₂. Results are shown as mean ± SD. #P < .05, ##P < .01 versus CON, **P < .01 versus H₂O₂.

SS-7 Reduced Cardiac Dysfunctions and Restored Histopathological Changes

SS-7 has been widely used in clinical practice to treat myocardial infarction in Inner Mongolia. In our previous study, we performed a pharmacodynamic evaluation of SS-7 on myocardial ischemia/reperfusion (I/R) injury in rats and found that 1.6 g/kg/d SS-7 was the best concentration. ¹⁴ As shown in Figure 3A, a flow chart of the cellular experiment is displayed. Rat cardiac functions were evaluated by echocardiography. Compared with IR rats, SS-7 significantly up-regulated the EF and FS values (Figure 3B, C, D). Relative to the heart of sham operated rat, HE staining revealed cardiomyocyte enlargement, cytoplasmic vacuole formation, and myofibrillar loss in I/R animals. Mongolian medicine shaosha-7 significantly decreased myocardial lesions, in rats (Figure 3E).

Figure 3.

SS-7 Improved Cardiac Dysfunctions and Restored Histopathological Changes. A: Schematic Diagram of Rats Treated with SS-7 or/and IR. B: Echocardiograms in Different Rat Groups. C: HE Staining of Myocardial Tissue of each rat Group. Results are Shown as Mean ± SD. #P < .05, ##P < .01 Versus CON, **P < .01 Versus IR.

Quantitative Identification of Differentially Expressed Proteins by TMT Based Proteomic Analysis

In order to globally understand the mechanisms of the cardioprotective effect of SS-7 on I/R rats, we conducted a TMT-based proteomic analysis. In our study, 4038 proteins and 32 792 Peptides were identified. Further analysis analyzed revealed that 348 (219 upregulated, 129 downregulated) proteins were identified in sham versus IR comparison, while 74 (59 upregulated, 15 downregulated) proteins were identified in the IR versus IR + SS-7 comparison (Figure 4). To select the target proteins related to SS-7, we applied a P-value threshold of <.05 for both the sham versus IR and IR versus IR + SS-7 comparisons. We found 66 DEPs associated with SS-7 and IR (S table 1).

Figure 4.

Protein Quantitative Results were Analyzed by a TMT-Based Quantitative Proteomics.

Bioinformatics Analysis

The biological function of SS-7 was identified through a Cluster of Orthologous Groups analysis, which was performed to functionally classify SS-7. The proteins were categorized according to biological process (BP), cellular components (CC), and molecular functions (MF). As shown in (Figure 5A), the differentially expressed proteins may be involved in the regulation of carbohydrate biosynthetic processes and the generation of precursor metabolites and energy. Our research also suggests that the starch and sucrose metabolism, as well as glycolysis/gluconeogenesis, are related to the treatment of SS-7 on IR (Figure 5D).

Figure 5.

Gene Ontology (GO) Analysis and KEGG Pathways of All Differentially Expressed Proteins.

SS-7 Alleviated H₂O₂ Induced Oxidative Stress Injury in Vitro

In our study, we found SS-7 could inhibit the intracellular ROS accumulation upon H₂O₂ treatment (Figure 6A). The increasing of ROS will further mediate membrane lipid peroxidation reaction and generate MDA. It also weakened the activities of antioxidant enzyme system SOD and GSH-Px. ²¹ During our results, SS-7 treatment significantly decreased the content of ROS and MDA in H9c2 cardiomyocytes with H₂O₂ induced cardiomyocytes injury. Meanwhile, SS-7 enhanced the activities of SOD and GSH-Px in H9c2 cardiomyocytes with H₂O₂ induced cardiomyocytes injury (Figure 6B). Taken together, these results suggested that SS-7 alleviated H₂O₂ -induced oxidative stress injury.

Figure 6.

SS-7 Decreased Cardiomyocyte Oxidative Stress in vitro. A: ROS was Evaluated Using the DCFH-DA Assay. B: The Levels of SOD, GSH-Px, and MDA were Measured by ELISA. Results are Shown as Mean ± SD. ##P < .01 Versus CON, *P < .05, **P < .01 Versus H₂O₂.

Validation of the Potential Targets and Pathways in IR rat Treatment with SS-7

In order to detect the effect of SS-7 on glucose metabolism, we used relevant kits to measure the levels of pyruvate and lactate, which are important products of glucose metabolism, in rat serum. We observed a significant accumulation of pyruvate and lactate in the serum of rats after ischemia-reperfusion (IR) compared to sham-operated rats, while SS-7 suppressed the accumulation of both pyruvate and lactate in the serum of IR rats (Figure 7A, B, D). PGK1 and AKR1B1 were chosen for further validation to better understand the molecular mechanisms underlying the effect of SS-7 against I/R or H₂O₂, using qPCR and immunohistochemistry. The results demonstrated that, compared with the sham group, the mRNA expression of PGK1 and AKR1B1 increased in the I/R group or H₂O₂ group, while SS-7 inhibited the expression of both PGK1 and AKR1B1 in I/R rats or H9c2 cardiomyocytes. In addition, the expression of PGK1 was detected using immunohistochemistry assay found that SS-7 reversed the expression of PGK1 in IR rats (Figure C).

Figure 7.

SS-7 Decreased Cardiomyocyte Glucose Metabolism. A: The Levels of Lactic Acid, and Pyruvate were Measured by ELISA. B: The Levels of PGK1 and AKR1B1 were Analyzed by qRT-PCR. C: PGK1 Staining by Immunohistochemistry. D: The Levels of PGKl and AKR1B1 in H9c2 Cell were Identified by qRT-PCR. Results are Shown as Mean ± SD. **P < .01 Versus CON, #P < .05, ##P < .01 Versus H₂O₂ or IR.

Discussion

Currently, there is still a lack of effective methods to treat I/R. ²² Mongolian medicine, as a form of traditional medicine, has been employed for the treatment of numerous challenging and intricate diseases, demonstrating its efficacy in alleviating and curing ailments. Our previous study showed that SS-7 exerted a cardio-protective effect in I/R rat. ¹⁴ Furthermore, the potential mechanism of SS-7 on I/R was investigated in vivo and in vitro in this research.

Nowadays, zebrafish have emerged as an ideal model organism in recent years and are widely used in the ecological safety evaluation of chemicals due to their sensitivity to harmful substances.^23,24 In this study, we used zebrafish embryos to assess the adverse impacts of SS-7. During the embryonic development stages, morphological development, heart rate, spontaneous tail coiling, and locomotive behavior are important indicators. Our results showed that SS-7 exposure had obvious sublethal effects at concentrations above 100 μg/mL. However, SS-7 exposure did not have a significant effect on the morphological development, heart rate, spontaneous tail coiling, and locomotive behavior within the range of 25-400 μg/mL. Based on the concentration of SS-7 in zebrafish, we analyzed its effects in vitro and in vivo. We found that SS-7 could effectively improve cardiac function, reverse myocardial pathological changes in I/R rats and protect cardiomyocytes. It is well known that the proteins are indispensable for maintaining vital activity, especially when organisms are exposed to endogenous or exogenous beneficial or harmful stimuli.^25–27

To further investigate the mechanism of SS-7 in the treatment of I/R, we used TMT-based proteomic analysis to explore the underlying mechanisms of SS-7 in treating IR. In our TMT-based proteomic analysis, we found that anti-I/R mechanism of SS-7 is closely related to glucose metabolism. It is well documented that the imbalance of glucose metabolism has become an attractive target for the treatment of AMI. ¹⁰ Accumulating studies disturbance of glucose metabolism is the fundamental energy metabolism disorder after AMI. ²⁸ Glucose metabolism plays a critical role in cardiomyocytes by providing energy and maintaining cellular function. ²⁹ However, myocardial ischemia-reperfusion disrupts energy metabolism and leads to dysregulation of glycolipid metabolism, ultimately leading to cell death and impaired cardiac function. ³⁰ A growing body of research has found that targeting g glucose metabolism can be effective against myocardial ischemia-reperfusion injury. ³¹ Disruption of glucose metabolism is one of the key determinants during myocardial ischemia-reperfusion injury. ³² Therefore, modulation and intervention of myocardial metabolism have emerged as promising avenues for the prevention and treatment of heart disease. To study the cardiac protective effect of SS-7 on glucose metabolism, we conducted preliminary research on glucose metabolism. The results showed that SS-7 could decreased pyruvate and lactate in rat serum, demonstrating a regulatory effect of SS-7 on glucose metabolism. Glucose metabolism is closely related to mitochondria, Oxidative stress occurs after mitochondrial damage. Oxidative stress is one of the most important pathological mechanisms of I/R injury. ³³ Oxidative stress refers to an imbalance between normal oxidant scavenging enzyme systems, including reactive oxygen species (ROS) production. ³⁴ It has reported that ROS can be generated during both the ischemia and reperfusion period. ³⁵ In our results, we found SS-7 inhibited the occurrence of oxidative stress.

As an essential enzyme, phosphoglycerate kinase 1 (PGK1) catalyzes the formation of ATP in the aerobic glycolysis pathway. ³⁶ It has been reported that PGK1 is predominantly elevated in tumor tissues, is related to the Warburg effects, and contributes to tumor growth and invasion. ³⁷ In the blood and synovium of patients with rheumatoid arthritis, PGK1 levels are increased. ³⁸ Recently, PGK1 has also been found in myocardial tissue, localized to infiltrating CD4T cells and Th17 cells. ³⁹ As a 36 kD monomeric protein, aldo-keto reductase family 1 member B1 (AKR1B1) is located in the cytoplasm. AKR1B1 converts aldehyde compounds to alcohol by consuming the H+ from nicotinamide adenine dinucleotide phosphate (NADPH), and participates in the reduction of toxic aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and its glutathione adducts (GS-HNE) in a physiological state. ⁴⁰ AKR1B1 increased AKR concentration activates glucose metabolism to sorbitol, finally resulting in an increased activation of inflammation and excessive cell apoptosis in a pathological conditions. ⁴¹ In this research, PGK1 and AKR1B1 were directly connected or has a strong relationship to the treatment of IR by SS-7 by immunohistochemistry and qPCR.

Conclusion

In conclusion, the present study discovered that SS-7 protects rats from I/R injury through PGK1/AKR1B1 mediated glucose metabolism. Our experimental results might provide a novel therapeutic target for I/R injury.

Limitations of the Study

In this paper, we found that PGK1/AKR1B1 mediated glucose metabolism was associated with SS-7 protects rats from IR injury, but whether PGK1/AKR1B1 is a definite target of SS-7 has not yet been clarified, and in the future, we will establish PGK1 or AKR1B1 knockout/overexpression mice, and then establish of the I/R injury model and intervene with SS-7, to clarify the target of SS-7's action on I/R injury.

Supplemental Material

sj-xlsx-1-npx-10.1177_1934578X251330718 - Supplemental material for Cardioprotective Effects of Shaosha-7 on Acute Myocardial Infarction Through Sucrose Metabolism Regulation

Supplemental material, sj-xlsx-1-npx-10.1177_1934578X251330718 for Cardioprotective Effects of Shaosha-7 on Acute Myocardial Infarction Through Sucrose Metabolism Regulation by Xiangjun Yang, Guohong Bao, Yuting Sun, Xiangwang Yu, Hongyang Wang and Guoyou Zhang in Natural Product Communications