AS-IV alleviates blocking of M3AchR induced myocardial apoptosis through p53/Akt signaling pathway under myocardial ischemia model

AS-IV alleviates blocking of M3AchR induced myocardial apoptosis through p53/Akt signaling pathway under myocardial ischemia model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article AS-IV alleviates blocking of M3AchR induced myocardial apoptosis through p53/Akt signaling pathway under myocardial ischemia model Chuqiao Shen, Shuo Chen, Fanjing Wang, Li Sun, Liang Yuan, Qiang Zuo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6667733/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Astragaloside IV (AS-IV), as the main active ingredient and nutritional adjunct of Astragali Radix, has a clear cardioprotective effect. However, it is unclear whether AS-IV could protect myocardium from ischemia by activating M3 subtype of muscarinic acetylcholine receptor (M3AchR) receptors and regulate cardiomyocytes apoptosis through p53/Akt. This study aims to establish an in vivo model of myocardial ischemia (MI) and utilize morphological, bioinformatics, and molecular biology methods to elucidate the mechanism by which AS-IV regulates MI and apoptosis through the M3AchR and p53/Akt pathway. The results suggested that AS-IV was able to alleviate the MI injury and aggravated apoptosis of cardiomyocytes caused by M3AchR inhibitors 4-DAMP in vivo. Moreover, AS-IV may have a protective effect on MI by directly acting on M3AchR. Mechanistically, AS-IV's anti-apoptotic effect could be associated with the regulation of the p53/Akt signaling pathway. Collectively, our research indicates that AS-IV could alleviate MI and exert myocardial protective effects by acting on the M3AchR and p53/Akt signaling pathways. This study provides a theoretical basis for exploring potential protective targets of AS-IV and elucidating new functions and mechanisms of AS-IV. Health sciences/Health care/Nutrition Health sciences/Cardiology Health sciences/Cardiology/Cardiovascular biology Health sciences/Diseases Health sciences/Diseases/Cardiovascular diseases Biological sciences/Drug discovery/Pharmacology/Receptor pharmacology Biological sciences/Drug discovery/Drug regulation Biological sciences/Chemical biology/Mechanism of action Biological sciences/Chemical biology/Pharmacology/Receptor pharmacology Astragaloside IV M3AchR p53/Akt apoptosis myocardial ischemia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Myocardial ischemia (MI) significantly contributes to the cardiovascular death rate globally. The onset of ischemic cardiomyopathy is often caused by insufficient myocardial oxygen supply due to narrowed coronary artery blockages. M3 subtype of muscarinic acetylcholine receptor (M3AchR) in cardiomyocytes plays an important role in myocardial contractile function 1 – 2 . Inhibiting M3AchR has promoted myocardial infarction 3 , fibrosis 4 – 5 , and other types of myocardial injury. Interestingly, the muscarinic receptor agonist has protected against myocardial injury from MI 6 , myocardial fibrosis 4 , hypotrophy 7 – 8 , and oxidative stress 9 via M3AchR. Acetylcholine (Ach), acting as a standard muscarinic receptor agonist, has the ability to protect the heart from myocardial injury through M3AchR 10 – 12 . However, the duration of action of Ach on M3AchR is too short and easily influenced by acetylcholinesterase. Therefore, exploring the mechanism and functional effects of supplementing food on M3AchR in cardiomyocytes has high research significance. Recent studies suggest that prolonged MI could lead to the apoptosis of cardiomyocytes. Cardiomyocyte apoptosis has also been described in M3AchR inhibitors-induced MI. Ach prevented myocardial apoptosis by activating the anti-apoptotic protein Bcl-2, whereas the M3AchR-specific inhibitor 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) showed a worsening effect 13 . The c-terminal tail of M3AchR has previously found to possess an anti-apoptotic function in cell research 14 . Evidence supports that blocking of M3AchR could exacerbate MI disease and hypoxic cardiomyocytes via the apoptosis signaling pathway 15 – 18 . Recently, p53 has been reported as a mediator in myocardial apoptosis 19 – 21 . Moreover, Akt is regulated by p53, further mediating cell apoptosis 22 – 23 . However, it is unknown how M3AchR regulate p53-induced myocardial apoptosis. Astragali Radix has been widely used as a functional food to protect cardiovascular, liver, and kidney diseases and improve health in traditional China or other East Asian countries. According to the U.S. Dietary Supplement Health and Education Act (DSHEA), it is categorized as a dietary supplement and consumed as an edible vegetable 24 . Astragali Radix has demonstrated protective properties against hyperlipidemia and myocardium offers benefits such as anti-inflammatory, antioxidation and lipid metabolism regulation 25 – 27 . Astragaloside IV (AS-IV), a triterpenoid saponin found in Astragali Radix, has significant potential as a dietary component for disease prevention 28 . In recent years, AS-IV has been consistently validated by numerous studies for its favorable myocardial protection effects 29 – 30 . It has been reported that administering AS-IV (25ng/ml/d) could effectively protect rat myocardial cells from hypoxia and apoptosis through the MAPK and Erk2 pathways 31 . Moreover, AS-IV has the ability to delay the progression of myocardial fibrosis, cardiac hypertrophy, and heart failure 32 – 33 . Research suggests that AS-IV exerts its protective effects on myocardial inflammation and oxidative stress by regulating TGF-β1 and Nrf-2 34–36 . Accordingly, AS-IV may protect myocardium from M3AchR inhibitors induced cardiomyocytes ischemia and apoptosis; however, the basic mechanisms remain unclear. We hypothesized that AS-IV could protect against myocardial apoptosis in a MI model by regulating the p53/Akt signaling pathway through M3AchR. This may provide a theoretical and experimental basis for elucidating the protective effect of AS-IV on MI through M3AchR receptors. 2. Methods 2.1 Animal model and treatment Adult male C57BL/6 male mice (age: 6–8 weeks) were purchased from Hangzhou Ziyuan Experimental Animal Technology Co. Ltd. (Hangzhou, China, Laboratory Animal Production). The experimental mice were raised under standard temperature (25 ± 2°C) and humidity (40–60%) and supplied with food, water, and adaptive feeding for 1 week. According to the literature 37 and the preliminary experimental results, the concentration of AS-IV (AS-IV) and the concentration of 4-DAMP and Ach 13 were selected, and all mice were randomly assigned to 6 groups (n = 6 for each group): (1) control group; (2) MI group; (3) MI + 4-DAMP (0.4µg/kg/d, ip) group; (4) MI + Ach (5mg/kg/d, ip) group; (5) MI + 4-DAMP + AS-IV (112mg/kg/d, ig) group; (6) MI + AS-IV group were selected in accordance with previous literature 13 . Pre-dose for 2 weeks. 2.2 MI mice model establishment The mice were anesthetized with 1% pentobarbital (40 mg/kg) injected during left thoracotomy. The left anterior descending (LAD) coronary artery was ligated with a 7–0 polypropylene suture, followed by skin suturing. Electrocardiogram (ECG) monitoring verified the success of MI surgery. T-wave changes and the ST-segment elevation in the ECG record confirmed that the mouse MI model was successfully established. All experimental protocols were approved by the Animal Experimentation Ethics Committee of the Anhui University of Chinese Medicine (AHUCM-mouse-2022009). All experiments were made to minimize animal suffering and conducted in accordance with guidelines and regulations. Our animal experimental research followed the in vivo experimental (ARRIVE) guideline 38 . Before harvesting the tissue, animals were anesthetized with pentobarbital sodium (40 mg/kg) injection. 2.3 Electrocardiography measurement The electrocardiogram of mice in each group was monitored one day after operation. After the mice were anesthetized with 1% pentobarbital (50mg/kg), the mice were placed on the mouse plate for fixation, and then the electrode needle was inserted into the corresponding position of the limbs in the way of the second lead to observe the electrocardiogram, and the changes in the heart rate of the electrocardiogram and the elevation level of the ST segment arch back were recorded. Each mouse was recorded three times for 120s each time. 2.3 Echocardiography measurement The small animal ultrasound imaging system (VINNO6LAB, Suzhou VINNO Technology Co., Ltd.) equipped with 18 MHz scanning connector was used to measure the echocardiography of mice. During the measurement, the mice were anesthetized with 1% pentobarbital (50mg/kg). Echocardiographic measurements were performed on left ventricular (LV) parasternal long-axis M-mode imaging. The mean value of the data is based on the measurement results of at least six cardiac cycles, including left ventricular end-systolic diameter (LVID), left ventricular end-diastolic diameter (LVIDd), and heart rate (HR). Left ventricular ejection fraction (EF) and left ventricular short axis shortening rate (FS) are calculated by VINNO6 software. 2.4 TTC staining for infarct area measurement The heart was carefully removed and rinsed with PBS buffer, and stored at -80°C for 20 min until frozen. Heart was then cutting into 5 equal thickness sections. The slices were placed in 1% 2,3,5-Triphenyltetrazolium Chloride (TTC) solution in a 37°C water bath for 15 min, followed by steeping in 10% neutralized formalin overnight. The heart slices were finally visualized using a Canon digital camera. The infarct area in the heart slices was presented in white, were calculated using Image J (1.53e) software. 2.5 Haematoxylin and eosin (HE) and Masson staining The hearts were fixed in 4% paraformaldehydefor 24 h. Next, ethanol at varying concentrations was employed for dehydration, and xylene was utilized for infiltration. Then, 2-mm ventricle tissue slices were embedded with paraffin and stained with hematoxylin and eosin (H&E) and Masson. The histopathological changes were observed under an optical microscope. 2.6 Tunel assay for myocardial apoptosis detection Tunel staining with 3,3-diaminobenzidine (DAB) was used to detect myocardial apoptosis in heart slices. According to the instructions provided with the kit, heart slices were treated with the Tunel detection solution (5 uL of TdT enzyme and 45 uL of Biotin-dUTP) and incubated at 37°C for an hour. Next, the slices were treated with 50 uL of the streptavidin–HRP working solution at room temperature for 30 minutes, then incubated with DAB and hematoxylin for 10 minutes at room temperature. The detection of cardiomyocyte apoptosis was performed with a Nikon eclipse 50i microscope. The normal cells appeared blue, while the Tunel-positive cardiomyocytes were brown. 2.7 Hoechst for myocardial apoptosis detection The Hoechst 33342/PI dual staining kit from Biosharp (BL116A) was utilized for Hoechst staining experiments. For fixed tissue samples, the fixative was removed following fixation, and the samples were washed 2–3 times with PBS buffer, each for 3–5 minutes. Subsequently, an appropriate volume of 5 µL Hoechst 33342 staining solution was directly applied to cover the samples, followed by the addition of 5 µL PI staining solution. Incubation was carried out at 4°C in the absence of light for 20 minutes. After removing the staining solutions, the samples were washed twice with PBS buffer, each for 3–5 minutes. Following mounting, the samples were observed directly under a fluorescence microscope. Hoechst 33342 utilizes ultraviolet light emitted by a krypton laser, with an excitation wavelength of 352 nm and an emission wavelength ranging from 400 to 500 nm, producing blue fluorescence. PI employs fluorescence induced by an argon ion laser, with an excitation wavelength of 488 nm and an emission wavelength exceeding 630 nm, generating red fluorescence. 2.8 Immunofluorescence study Slices of heart tissue were subjected to microwave antigen repair, blocked, and then incubated with a commercial primary anti-M3AchR (cat. no. AMR-006; 1:200; Thermo), anti-Akt (cat. no. AF6261, 1:200; Affinity Biosciences), anti-phosphorylated (anti-p)-Akt (cat. no. AF0016; 1:200; Affinity Biosciences) for 4°C overnight. After washing the heart slices three times with 0.01 M TBS (pH 7.4) for 5 minutes each, they were incubated at 37°C with TRITC-conjugated goat anti-rabbit IgG (cat. no. 139931; ZSGB-BIO, Beijing China) for 50 minutes. The nuclei were stained using a 4′,6-diamidino-2-phenylindole (DAPI; cat. no. G1012; Servicebio) solution in darkness for 5 minutes. The slides were finally imaged with a NIKON ECLIPSE C1 microscope, and the immunofluorescence was examined using Image J (1.53e) software. 2.9 Western blotting The total proteins derived from C57 mouse hearts underwent fractionation with the RIPA cell lysate (containing 1-mm phenylmethylsulfonyl fluoride [PMSF]) and added to 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). Then, the proteins were transferred into the polyvinylidene fluoride (PVDF) membrane. The membranes were incubated with primary anti-Bcl-2 (1:1,000; cat. no. ab32124; Abcam), anti-Bax (cat. no. ab32503; 1:1,000; Abcam), anti-caspase-3 (cat. no. ab32351; 1:5,000; Abcam), anti-cleaved-caspase-3 (cat. no. 341034; 1:1,000; ZENBIO), anti-Akt (cat. no. AF6261; 1:1,000; Affinity Biosciences), anti-phosphorylated (anti-p)-Akt (cat. no. AF0016; 1:1,000; Affinity Biosciences), anti-hypoxiainducible factor (HIF)-1α (cat. no. AF1009; 1:500; Affinity Biosciences), anti-M3AchR (cat. no. PA5-85322; 1: 1,000; Thermo), anti-p53 (cat. no. AF0879; 1:1,000; Affinity Biosciences), anti-phosphorylated (anti-p)-p53 (cat. no. R380837; 1:500; ZENBIO), and β-actin (Zs-BIO) at 4°C overnight. Subsequently, the blotted membranes underwent 2 h of incubation with the horseradish peroxidase (HRP)-conjugated secondary antibodies at ambient temperature. Enhanced chemiluminescence (ECL) substrates (Millipore) contributed to the visualization of the signals. 2.10. Detection of CK-MB, cTnI and LDH in serum Serum levels of CK-MB (cat. no. RX201007M; Quanzhou Ruixin Biological Technology Co., Ltd.; Quanzhou; China), cTnI (cat. no. H149-2-2; Nanjing Jiancheng Bioengineering Institute; Nanjing; China), and LDH (cat. no. A020-2-2; Nanjing Jiancheng Bioengineering Institute; Nanjing; China) were measured by experimental kits with a RT-6100 microplate analyzer (Rayto Life and Analytical Sciences Co.,Ltd.) according to the manufacturer’s instructions. 2.11 Statistical analysis All results are presented as the mean ± standard deviation (SD). Student’s t -test was performed for comparisons between the two groups. One-way ANOVA analysis was performed for > 2 groups with a post-hoc least significant difference (LSD) test to evaluate the significant differences between each group. Statistical data were analyzed with the SPSS 22.0 software. P < 0.05 was considered to indicate statistical significance. 3. Results 3.1 Effect of AS-IV on cardiac function of MI model through M3 receptor We used parasternal left ventricular long-axis imaging to evaluate the cardiac function of mice in each group, and recorded the changes of left ventricular ejection fraction (EF), left ventricular short-axis shortening rate (FS), left ventricular end-diastolic diameter (LVIDd) and left ventricular systolic diameter (LVIDs), and compared the functional differences between the groups (Fig. 1 A). The results showed that compared with the MI model group, the 4-DAMP group significantly reduced the left ventricular ejection fraction (EF) ( P < 0.01; Fig. 1 B(a)) and short-axis shortening rate (FS) ( P < 0.01; Fig. 1 B(b)) of mice, increased the left ventricular end-diastolic diameter (LVIDd) ( P < 0.01; Fig. 1 B(c)) and systolic diameter (LVIDs) ( P < 0.01; Fig. 1 B(d)) of mice with myocardial infarction, while the AS-IV group significantly weakened the above-mentioned effects of the 4-DAMP group. Compared with 4-DAMP + AS-IV group, AS-IV group significantly increased the left ventricular ejection fraction (EF) ( P < 0.01; Fig. 1 B(a)) and short-axis shortening rate (FS) ( P < 0.01; Fig. 1 B(b)) of mice, and decreased the left ventricular end-diastolic diameter (LVIDd) ( P < 0.01; Fig. 1 B(c)) and systolic diameter (LVIDs) ( P < 0.01; Fig. 1 B(d)) of mice with myocardial infarction, the difference was statistically significant. 3.2 AS-IV exerts a protective effect on myocardial injury by activating M3AchR in mice myocardial ischemia model By observing the ST segment and T wave (Fig. 2 A), we could find that compared with the control group, the ST segment and T wave of MI group and MI + 4-DAMP group were significantly elevated, indicating that the heart had ischemia; MI + Ach and MI + AS-IV groups could significantly reverse the pathological state of ST segment and T wave in model mice. Compared with the MI + 4-DAMP group, the ST segment and T wave of the MI + 4-DAMP + AS-IV group decreased slightly. The results showed that AS-IV could alleviate the abnormal elevation of ST segment in mice with myocardial ischemia induced by M3 receptor. The results of ECG heart rate showed that the heart rate of MI group was significantly lower than that of Control group ( P < 0.01; Fig. 2 C); Compared with MI group, the heart rate of 4-DAMP group was significantly higher, that of Ach group was significantly lower, and that of AS-IV group was significantly lower ( P < 0.01; Fig. 2 C); Compared with 4-DAMP group, the heart rate of 4-DAMP + AS-IV group was significantly lower ( P < 0.01; Fig. 2 C); Compared with 4-DAMP + AS-IV group, the heart rate of AS-IV group was significantly lower ( P < 0.01; Fig. 2 C). In the assessment of myocardial infarct size (Fig. 2 B), we observed that the 4-DAMP group significantly increased the myocardial infarct size in mice compared to the MI model group ( P < 0.01; Fig. 2 D). However, after administration of AS-IV or the M3 receptor agonist ACh, the myocardial infarct size significantly decreased ( P < 0.01; Fig. 2 D). Compared to the 4-DAMP + AS-IV group, the AS-IV group significantly reduced the myocardial infarct size in mice, with a statistically significant difference ( P < 0.01; Fig. 2 D). 3.3 Effect of AS-IV on myocardial injury indicators and pathological changes in a myocardial ischemia model via the M3 receptor This study aimed to explore the anti-M3AchR effect on myocardial injury in vivo . After a week of pretreatment with 4-DAMP and Ach, the MI model for drug administration in each group was formed. When compared to the MI group, 4-DAMP exacerbated MI morphological changes including myocardial myolysis and inflammatory cells infiltration, and myocardial apoptosis staining with brownish yellow nuclei, while 4-DAMP + AS-IV improved 4-DAMP morphological changes and myocardial apoptosis on histological H&E staining assay (Fig. 3 A). Masson's trichrome staining was performed to detect fibrosis deposition in myocardium. Fibrous tissue stained blue and myocardial fibers stained red. Collagen content and disarray of myocardial fibers was significantly increased in the 4-DAMP group compared with the MI group (Fig. 3 B). Compared with the 4-DAMP group, the collagen content and myocardial fiber disorder in the 4-DAMP + AS-IV group were significantly reduced. The LDH activity ( P < 0.01, Fig. 3 C), CK-MB ( P < 0.01, Fig. 3 C), and cTnI ( P < 0.01, Fig. 3 C) concentrations in 4-DAMP group plasma were significantly elevated than the MI group, while the 4-DAMP + AS-IV group lactate dehydrogenase (LDH) activity ( P < 0.01, Fig. 3 C), CK-MB ( P < 0.01, Fig. 3 C), and cTnI ( P < 0.05, Fig. 3 C) concentrations were decreased compared to the 4-DAMP group. 3.4 Effect of AS-IV on myocardial apoptosis in MI model via M3 receptor The Tunel (DAB) and Hochest staining results indicate that, compared to the MI myocardial infarction model group, administration of the M3 receptor inhibitor 4-DAMP (0.4 mg/kg) significantly increased cardiomyocyte apoptosis in the myocardial tissue of ischemic mice ( P < 0.01, Fig. 4 A,B), suggesting that inhibiting the M3 receptor could enhance cardiomyocyte apoptosis. Compared to the MI myocardial infarction model group, the AS-IV group reduced cardiomyocyte apoptosis in ischemic mice ( P < 0.01, Fig. 4 A,B). Compared to the 4-DAMP group, the combination of 4-DAMP and AS-IV attenuated the inhibitory effect of AS-IV on cardiomyocyte apoptosis in ischemic mice, indicating that AS-IV affects cardiomyocyte apoptosis in ischemic mice by acting on the M3 receptor ( P < 0.01, Fig. 4 A,B). 3.5 AS-IV could significantly affect the expression of M3AChR in MI mouse model Western-Blot and immunofluorescence staining results showed that administration of M3 receptor inhibitor 4-DAMP (0.4 mg/kg) significantly reduced the expression of M3AChR in myocardial tissues of mice with myocardial ischemia compared with the MI model group, suggesting that inhibition of M3AChR could reduce the expression of M3AChR ( P < 0.05, P < 0.01, Fig. 5 A,B). Compared with the MI model group, the AS-IV group significantly elevated the expression of M3 receptors in the myocardial tissues of mice with myocardial ischemia ( P < 0.05, P < 0.01, Fig. 5 A,B). Compared with the 4-DAMP group, the combination of 4-DAMP + AS-IV attenuated the effect of 4-DAMP on the decrease of M3AChR expression in the myocardial tissues of mice with myocardial ischemia ( P < 0.01, Fig. 5 A,B). 3.6 Analysis of the Binding Targets of AS-IV with M3AChR Protein As depicted in the Fig. 6 , the analysis conducted using the CB-Dock software indicates that there exist two binding pockets (Pocket1 and Pocket2) between AS-IV, serving as the ligand, and the M3AChR protein. The FitDock Score for these two pockets is -3.5 and 29, respectively, with the templates being t2:4j4q and t5:7bu6 (Table 1 ). In addition, there are five structural molecular binding sites (Pocket 3–7) between AS-IV, acting as the ligand, and the M3AChR protein. The Vina Score for these sites is -9.1, -8.7, -7.8, -7.4, and − 7.3, respectively (Table 2 ). The results of the analysis predict that AS-IV, acting as a ligand, exhibits favorable binding characteristics with the M3AChR protein. Table 1 Templete based docking results. Pockets FitDock Score Template ID Template 1 -3.5 t2 4j4q 2 29.3 t5 7bu6 Table 2 Structure based docking results. Pockets Vina Score Cavity Size Center X Y Z 3 -9.1 5430 -9 -7 51 4 -8.7 1974 -17 7 -32 5 -7.8 1814 8 4 61 6 -7.4 2657 12 7 25 7 -7.3 2145 15 17 39 3.7 Identification of hub proteins and signaling pathways for the treatment of MI by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment As Fig. 7 shown, we obtained gene targets related to myocardial hypoxia from GeneCards ( https://www.genecards.org/,v5.3.0 ); Online Mendelian Inheritance in Man (OMIM, http://www.omim.org/ , Updated June 26, 2021); Therapeutic Target Database (TTD, http://db.idrblab.net/ttd/ , Updated June 1, 2020), and Drugbank database ( https://go.drugbank.com/ , version 5.1. 8). After the results of KEGG pathway top 20 pathway ranking were analyzed, it was concluded that myocardial hypoxia is closely related to HIF-1α and apoptosis pathway (Fig. 7 A). From the analysis of the results of p53 pathway map in KEGG, it could be concluded that under hypoxic myocardium, the differentially expressed genes were mainly enriched in the pathways related to Bcl-2, Caspase-3 and PAI. And the results of gene enrichment pathway related to Bcl-2 and Caspase-3 pointed to apoptosis (Fig. 7 B). 3.8 AS-IV regulated the expression of HIF-1α through M3AChR under MI model As shown in Fig. 8 , the administration of the M3AChR inhibitor 4-DAMP (0.4 mg/kg) was able to significantly increase the expression of HIF-1α in myocardial tissues of mice with myocardial ischemia compared with the MI infarction model group, suggesting that inhibition of the M3AChR was able to increase the phenomenon of posthypoxia in myocardial ischemia ( P < 0.01, Fig. 8 ). Compared with the MI infarction model group, the AS-IV group significantly decreased the expression of HIF-1α in the myocardial tissues of myocardial ischemic mice ( P < 0.01, Fig. 8 ). Compared with the 4-DAMP group, the combination of 4-DAMP + AS-IV attenuated the increase of HIF-1α expression in the myocardial tissues of myocardial ischemic mice by 4-DAMP ( P < 0.01, Fig. 8 ). 3.9 AS-IV could act on M3AChR to reduce the expression of p-p53/p53 and p-Akt/Akt and thereby improving MI Western-Blot and immunofluorescence results showed that 4-DAMP (0.4 mg/kg), an M3AChR inhibitor, significantly increased the expression of p-p53/p53 and decreased the expression of p-Akt/Akt in myocardial tissues of mice with myocardial ischemia compared with the MI infarction model group ( P < 0.01, Fig. 9 ), suggesting that the inhibition of M3AChR could increase the expression of p-p53/p53 and decreased the expression of p-Akt/Akt. Compared with the MI infarction model group, the AS-IV group significantly reduced the expression of p-p53/p53 and increased the expression of p-Akt/Akt in the myocardial tissues of mice with myocardial ischemia ( P < 0.01, Fig. 9 ). Compared with the 4-DAMP group, the combination of 4-DAMP + AS-IV attenuated the reduction of p-p53/p53 and increase of p-Akt/Akt expression in the myocardial tissues of mice with myocardial ischemia ( P < 0.01, Fig. 9 ). 3.10 Effect of AS-IV on apoptosis rate and apoptotic proteins Bax, Bcl-2 and Caspase-3 in MI model via M3AChR Western-Blot results showed that compared with the MI group, administration of the M3AChR inhibitor 4-DAMP (10 µM) was able to significantly increase the protein expression of Bax, Caspase-3 and cleaved-Caspase-3 and decrease the protein expression of Bcl-2 in hypoxic myocardium ( P < 0.01, Fig. 10 ). It is suggested that inhibition of M3AChR could play a pro-apoptotic role by activating the protein expression of Bax, Caspase-3 and cleaved-Caspase-3 and inhibiting the protein expression of Bcl-2. Compared with the MI group, the AS-IV group decreased the expression of Bax, Caspase-3 and cleaved-Caspase-3 and elevated the expression of Bcl-2 in hypoxic myocardium ( P < 0.01, Fig. 10 ). Compared with the 4-DAMP group, the combination of 4-DAMP + AS-IV attenuated the decreasing effect of AS-IV on the expression of Bax, Caspase-3 and cleaved-Caspase-3 and the elevating effect on the expression of Bcl-2 in hypoxic myocardium ( P < 0.01, Fig. 10 ). 4. Discussion In recent years, the regulation of cardiomyocytes function by the M3AchR has garnered widespread attention within the cardiovascular research community 39 – 40 . Studies have shown that the M3AchR inhibitor 4-DAMP could significantly increase the effects of myocardial injury and apoptosis after hypoxia 8 , 10 , while the M3AchR agonist Ach could protect against myocardial injury and apoptosis after ischemia 41 – 42 . AS-IV, as the primary active ingredient in Astragali Radix, exhibits well-defined myocardial protection after ischemia. Therefore, this study first investigates whether AS-IV protects the symptoms of MI through M3AchR. In this research, we replicated a mouse MI model by ligating the left anterior descending branch (LAD), and observed changes in the mouse heart through electrocardiogram, TTC staining, serum biomarkers and pathological experiments. To investigate whether AS-IV affects cardiac function and myocardial injury after MI through the M3AchR, we used specific M3AchR inhibitors 4-DAMP and agonists Ach to observe and study the effects of AS-IV on the regulation of M3AchR in MI mice. The results indicated that administration of M3AchR inhibitor 4-DAMP could mitigate cardiac function in mice with MI, exacerbate myocardial injury and fibrosis after ischemia; AS-IV could protect against ischemia induced myocardial injury and fibrosis exacerbation caused by 4-DAMP, and improve cardiac function in the body in vivo . This study suggests that AS-IV improves cardiac function in MI mice and has a protective effect on myocardial injury and fibrosis after ischemia, which is associated with agonizing the M3AchR. Thus, AS-IV could be a beneficial dietary option or combination to ease MI symptoms through M3AchR, potentially broadening its application scope in everyday health practices. Notably, it is necessary to understand how AS-IV acts on M3AchR to exert MI protection. This experiment first demonstrated that AS-IV could improve the downregulation of M3 receptor expression by ischemia. This aligns with earlier findings that enhancing M3AchR expression might avert cardiac death due to MI 43 . Moreover, our analysis further revealed seven highly probable drug targets for AS-IV to interact with the M3AchR. To delve deeper into the mechanism of AS-IV's myocardial protective action, we employed CB-Dock (Cavity-detection Guided Blind Docking) to analyze the low-molecular-weight protein interactions between AS-IV and the M3AchR. Through CB-Dock analysis, we identified potential molecular targets for AS-IV with the M3AchR. This suggests that AS-IV may exert its MI protective effects by directly act on M3AchR. The M3AchR inhibitor effect promoting MI is supposed to correlate with apoptosis in cardiomyocytes. However, the mechanism of myocardial apoptosis caused by M3AchR inhibitor induced MI injury has not been elucidated yet. Therefore, exploring the protective mechanism of M3AchR against MI provides a preliminary basis for demonstrating the mechanistic impact of AS-IV on M3AchR. The pathway triggered by MI that leads to cell apoptosis could be mediated by central mediator changes to Bax/Bcl-2 and caspase-3 activation, which is regulated by the p53 protein 44 – 46 , as well as the involvement of HIF-1α upregulation 13 – 14 . Through KEGG pathway analysis, we found a strong correlation between myocardial ischemic injury and the apoptotic pathway. Our research results indicate that inhibiting M3AchR increases the expression of HIF-1α, Bax, and cleaved Caspase-3/Caspase-3, while reducing the expression of Bcl-2, exacerbating the apoptotic changes of ischemic myocardium. Phosphorylated Akt, activated by phosphatidylinositol-3 kinase, is a vital mediator in cell survival and the anti-apoptotic process 46 – 47 . Moreover, p53 activation could alleviate the Akt protein expression level in the mutant p53 mouse model 49 and implicated in the cardiomyocytes proliferation and apoptosis 50 – 52 . The results showed that M3AchR inhibitor-treated ischemic myocardium promoted p-p53/p53 expression and repressed p-Akt/Akt expression. Cumulatively, inhibition of M3AchR could exacerbate apoptosis in ischemic myocardium via regulating p53 expression and Akt phosphorylation. Finally, we investigated the regulation of apoptosis and p53/Akt related apoptotic protein expression in mice with MI by AS-IV through M3AchR. The results show that inhibiting the M3AchR increases the expression of HIF-1α protein, while AS-IV decreases its expression through the M3AchR, suggesting a significant correlation between the regulation of M3AchR and AS-IV's protective effects with the degree of MI. By employing M3AchR inhibitors, we found that AS-IV reduced myocardial apoptosis in ischemic mice through M3AchR. Subsequently, Western-Blot results indicate that AS-IV increases Bax and cleaved-Caspase-3/Caspase-3 expression and decreases Bcl-2 expression in MI through the M3AchR. We measured p-p53/p53 and the downstream p-Akt/Akt levels to elucidate how the AS-IV regulates p53- and Akt-induced apoptosis in cardiomyocytes under MI through M3AchR. Inhibiting the M3AchR enhances p-p53/p53 protein expression, while AS-IV decreases its expression through the M3AchR. Moreover, inhibiting M3AchR could reduce the expression of p-Akt/Akt in myocardium, while AS-IV increases p-Akt/Akt through M3AchR. The findings of this study indicate that p53 and Akt is an important link in AS-IV's mechanism for inhibiting myocardial apoptosis. Therefore, AS-IV could act on M3AchR to alleviate MI symptoms, improve heart function and myocardial injury in mice, and inhibit myocardial apoptosis in ischemic mice through the p53/Akt pathway. In summary, this study is the first to apply the M3AchR to investigate the mechanism of AS-IV in inhibiting myocardial apoptosis under MI model. By using morphological, bioinformatics, and molecular biology methods, our research revealed that AS-IV could attenuate the ischemic injury and apoptosis aggravation of cardiomyocytes by M3AchR inhibitors in vivo. Moreover, the anti-apoptotic effect of AS-IV might relate to the regulation of p53/Akt signaling pathway (Fig. 11 ). However, further research is needed to verify whether AS-IV could directly interact with M3AchR or have other forms of impact. This study provides new experimental evidence to reveal the cardioprotective effect of AS-IV through M3AchR and p53/Akt signaling pathway. Crucially, this research sets a foundation for the application of AS-IV as a nutritional adjunct to relieve MI symptoms. Declarations ASSOCIATED CONTENT Data Availability Statement Available from the corresponding author on reasonable request. DECLARATION OF INTERESTS The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. AUTHOR INFORMATION Corresponding Authors Yixuan Lin - Department of Endocrinology, The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui, 230031, China; Phone: +8615256595986; Email: [email protected] Qiang Zuo - Department of Cardiology, The First Affiliated Hospital, Anhui University of Chinese Medicine, Hefei, Anhui, 230031, China; Phone: +8613605691870; Email: [email protected] Author Contributions CS, QZ and YL designed the research and revised the manuscript. CS, SC, FW, LS, LY and QZ conducted the experiments, and involved in the data acquisition, and manuscript writing. CS, FW and QZ analyzed and interpreted the data. All authors have read and approved the final manuscript. YL and QZ confirm the authenticity of all the raw data. ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (no. 81904147), the Youth Research Project of Health Commission of Anhui Province (grant no. AHWJ2023A30241), the Scientific Research Foundation of Education Department of Anhui Province of China (grant no. 2024AH051007), the Scientific Research Foundation of Education Department of Anhui Province of China (grant no. 2023AH050867). DATA AVAILABILITY Data is provided within the manuscript. References Sassu, E.; Tumlinson, G.; Stefanovska, D.; Fernandez, M. C.; Iaconianni, P.; Madl, J.; Brennan, T. A.; Koch, M.; Cameron, B. A.; Preissl, S.; Ravens, U.; Schneider-Warme, F.; Kohl, P.; Zgierski-Johnston, C. M.; Hortells, L., Age-related structural and functional changes of the intracardiac nervous system. J Mol Cell Cardiol 2024, 187 , 1-14. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6667733","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":477804694,"identity":"4944cc1a-3336-4c2e-8de8-9ddce4a42645","order_by":0,"name":"Chuqiao Shen","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chuqiao","middleName":"","lastName":"Shen","suffix":""},{"id":477804695,"identity":"9695bcc8-8dfe-473a-a8d7-af3fcbdcd14b","order_by":1,"name":"Shuo Chen","email":"","orcid":"","institution":"Ministry of Education, Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Chen","suffix":""},{"id":477804696,"identity":"05b83233-b4b0-439c-b83e-83e91d76e97d","order_by":2,"name":"Fanjing Wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fanjing","middleName":"","lastName":"Wang","suffix":""},{"id":477804697,"identity":"ee440fd1-65a6-4507-9f95-391c8a81c334","order_by":3,"name":"Li Sun","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Sun","suffix":""},{"id":477804698,"identity":"7f8c4754-f936-4959-b082-415beea9c80c","order_by":4,"name":"Liang Yuan","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Yuan","suffix":""},{"id":477804699,"identity":"d878f9fe-3cd6-424e-ad61-dc955e5f8e91","order_by":5,"name":"Qiang Zuo","email":"","orcid":"","institution":"Anhui University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Zuo","suffix":""},{"id":477804700,"identity":"b585309b-decc-43a8-a5af-082710ffa7ce","order_by":6,"name":"Yixuan Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBAC+/PNBw58+FFTz8/eQKyeG8cSH87sOZYg2XOAWC0HcoyNediYEwxuJBCpg7HhjJk0Dw9bnuTMxxtvMNTYRBPUwszcViY5x0KmmF86rdiC4VhabgMhLWwMh7dJvOFhY5w5O8dMgrHhMGEtPAwJZhJAvzBuuHmGSC0SDCnGhkAtiRtu8BCpxUACEsjGkj1AvyQQ4xcDfkhUyvGzH95440ONDWEtqDYmkKIcooVUHaNgFIyCUTAyAADUSkHBRXf5GQAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Anhui University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yixuan","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2025-05-15 01:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6667733/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6667733/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85652970,"identity":"387de3bb-d6e1-4b5a-96b8-46b0116cb40f","added_by":"auto","created_at":"2025-06-30 09:50:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106189,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of AS-IV on cardiac function after myocardial ischemia in mice through M3 receptor interference. (A) Ultrasound images of mouse hearts in each group. (B) Ultrasound results of mouse heart. (a) The percentage of ejection fraction. (b) The percentage of fraction shortening. (c) Left ventricular internal diameter at end-diastole (LVIDd). (d) Left ventricular internal diameter at end-systole (LVIDs). \u003csup\u003e\u003cem\u003e\u0026nbsp;*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe Control group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe MI group; \u003csup\u003e\u003cem\u003e^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e^^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP group; \u003csup\u003e\u003cem\u003e\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP+AS-IV group. Data are presented as the mean ± SD (n=6).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/9ad86fefa6e31942db92a33d.jpg"},{"id":85652971,"identity":"79e6be03-f651-4b13-a5ea-9b1f7479c3c6","added_by":"auto","created_at":"2025-06-30 09:50:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129189,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effects of AS-IV on ECG of MI rats through M3 receptor. (n=6). (B) Effects of AS-IV on MI infarct area in mice through M3 receptor (Scale bar = 5 mm). (n=3). (C) Heart rate of electrocardiogram. (n=6). (D) The infarct area ratio in mice. (n=3). \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe Control group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe MI group; \u003csup\u003e\u003cem\u003e^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e^^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP group; \u003csup\u003e\u003cem\u003e\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP+AS-IV group. Data are presented as the mean ± SD (n=6).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/5f0030fcdbb31c620dee361e.jpg"},{"id":85653750,"identity":"0e6e234b-6772-440c-a516-ccd84b9e910f","added_by":"auto","created_at":"2025-06-30 09:58:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":234517,"visible":true,"origin":"","legend":"\u003cp\u003eThe mechanism of M3 receptor inhibition induced cardiomyocyte apoptosis in HL-1 cells exposed to hypoxia. The histological pathology effects of AS-IV on myocardium of MI mice through M3 receptor. (A) The heart section of myocardial ischemia with hematoxylin-eosin staining (×200, Scale bar = 50 μm; ×400, Scale bar = 25 μm) in the experimental groups (n = 3). (B) The heart section of myocardial ischemia with Masson-trichrome staining (×200, Scale bar = 50 μm; ×400, Scale bar = 25 μm) in the experimental groups (n = 3). (C) Creatine phosphokinase (CK)-MB, Cardiac troponin (cTn) I concentration and Lactate dehydrogenase (LDH) activity in the mice plasma (n = 6). \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e the control group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe hypoxia group.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/7e410fd4c462212dd51d08b4.jpg"},{"id":85653929,"identity":"5e803e96-04b5-4aee-a432-03d685115644","added_by":"auto","created_at":"2025-06-30 10:06:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":128265,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Tunel staining (×200, Scale bar = 50 μm, Tunel-positive: black arrow) in the experimental groups (n=3). (B) Comparison of the apoptosis rates of myocardium showed by Hoechst 33342/PI staining (×200, Scale bar = 50 μm) in the experimental groups (n = 3).\u003csup\u003e\u003cem\u003e *\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e the control group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe hypoxia group.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/19575071265a641aa3e702fa.jpg"},{"id":85653756,"identity":"8d4e8a91-2064-41a7-ba67-e2f0c19c6d8e","added_by":"auto","created_at":"2025-06-30 09:58:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":126355,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of AS-IV on M3AChR expression with M3 receptor interference under MI mouse model. (A) The protein level of M3AChR was detected by western blot. (B) Representative immunofluorescence staining images of M3AChR in ventricular sections (×200, Scale bar = 50 μm). Data are presented as the mean ± SD (n = 3).\u003csup\u003e\u003cem\u003e *\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe Control group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe MI group; \u003csup\u003e\u003cem\u003e^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e^^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP group; \u003csup\u003e\u003cem\u003e\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP+AS-IV group.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/c0d92dd88adef9b5e2e3dd36.jpg"},{"id":85652986,"identity":"c23502c9-1b51-4393-a67e-21d7903e0828","added_by":"auto","created_at":"2025-06-30 09:50:59","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":113972,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking validation of AS-IV binding sites with M3AChR protein. The top 7 of the molecular binding abilities (residues in red boxes are active site residues).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/09b36a2018aca6d71a1931dd.jpg"},{"id":85652974,"identity":"78254853-de50-4915-97ff-d70b13ed8d91","added_by":"auto","created_at":"2025-06-30 09:50:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":135022,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Enrichment of KEGG pathway of differentially expressed genes related to myocardial hypoxia. The top 20 of KEGG pathways were presented on the diagram. (B) Enrichment of p53-related signaling pathways of differentially expressed genes related to myocardial hypoxia (Red star indicates gene enrichment point).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/cacb5f427a5e3615741df763.jpg"},{"id":85652975,"identity":"9fcc0783-3f48-4b30-add5-c9abc5fbb358","added_by":"auto","created_at":"2025-06-30 09:50:59","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":33674,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of AS-IV on HIF-1α expression with M3AChR interference under MI model. Data are presented as the mean ± SD (n=3).\u003csup\u003e\u003cem\u003e *\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe Control group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe MI group; \u003csup\u003e\u003cem\u003e^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e^^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP group; \u003csup\u003e\u003cem\u003e\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP+AS-IV group.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/5aa8895d38fda164506c92c1.jpg"},{"id":85653931,"identity":"3af6d002-c9d5-44f7-908e-93fa164e388b","added_by":"auto","created_at":"2025-06-30 10:06:59","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":105451,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of AS-IV on p-p53/p53 and p-Akt/Akt expression with M3 receptor interference under myocardial ischemic model. (A) Relative protein expression of p-p53, p53, p-Akt and Akt. (B) Immunofluorescence staining of p-Akt and Akt. Data are presented as the mean ± SD (n=3). *P \u0026lt; 0.05 and **P \u0026lt; 0.01 vs. the Control group; #P \u0026lt; 0.05 and ##P \u0026lt; 0.01 vs. the MI group; ^P \u0026lt; 0.05 and ^^P \u0026lt; 0.01 vs. the 4-DAMP group; \u0026amp;P \u0026lt; 0.05 and \u0026amp;\u0026amp;P \u0026lt; 0.01 vs. the 4-DAMP+AS-IV group.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/8a0d3e97b35e69a07c65ad88.jpg"},{"id":85653754,"identity":"e4f8d1f5-996d-40a0-bcd7-ae1e82cf4b9f","added_by":"auto","created_at":"2025-06-30 09:58:59","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":74174,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of AS-IV on Bax, Bcl-2 and Caspase-3 expression with M3 receptor interference under MI model. Data are presented as the mean ± SD (n=3).\u003csup\u003e\u003cem\u003e *\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe Control group; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe MI group; \u003csup\u003e\u003cem\u003e^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e^^\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP group; \u003csup\u003e\u003cem\u003e\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs. \u003c/em\u003ethe 4-DAMP+AS-IV group.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/48b237dd47e7403505788125.jpg"},{"id":85652987,"identity":"01147591-be07-4b6d-9f8e-70407badd7fe","added_by":"auto","created_at":"2025-06-30 09:50:59","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":55360,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of AS-IV's effect on reducing myocardial apoptosis triggered by M3AchR obstruction.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/ca500076493516ea92bf7658.jpg"},{"id":86709555,"identity":"1dfbbdcb-93fe-499f-967f-63ca758bdeea","added_by":"auto","created_at":"2025-07-14 18:08:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2661092,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6667733/v1/a942d82a-af9b-4828-b5f5-a56365e5a0b3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"AS-IV alleviates blocking of M3AchR induced myocardial apoptosis through p53/Akt signaling pathway under myocardial ischemia model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMyocardial ischemia (MI) significantly contributes to the cardiovascular death rate globally. The onset of ischemic cardiomyopathy is often caused by insufficient myocardial oxygen supply due to narrowed coronary artery blockages. M3 subtype of muscarinic acetylcholine receptor (M3AchR) in cardiomyocytes plays an important role in myocardial contractile function\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Inhibiting M3AchR has promoted myocardial infarction \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, fibrosis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, and other types of myocardial injury. Interestingly, the muscarinic receptor agonist has protected against myocardial injury from MI \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, myocardial fibrosis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, hypotrophy \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and oxidative stress \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e via M3AchR. Acetylcholine (Ach), acting as a standard muscarinic receptor agonist, has the ability to protect the heart from myocardial injury through M3AchR \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, the duration of action of Ach on M3AchR is too short and easily influenced by acetylcholinesterase. Therefore, exploring the mechanism and functional effects of supplementing food on M3AchR in cardiomyocytes has high research significance.\u003c/p\u003e \u003cp\u003eRecent studies suggest that prolonged MI could lead to the apoptosis of cardiomyocytes. Cardiomyocyte apoptosis has also been described in M3AchR inhibitors-induced MI. Ach prevented myocardial apoptosis by activating the anti-apoptotic protein Bcl-2, whereas the M3AchR-specific inhibitor 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) showed a worsening effect \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The c-terminal tail of M3AchR has previously found to possess an anti-apoptotic function in cell research \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Evidence supports that blocking of M3AchR could exacerbate MI disease and hypoxic cardiomyocytes via the apoptosis signaling pathway\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Recently, p53 has been reported as a mediator in myocardial apoptosis \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Moreover, Akt is regulated by p53, further mediating cell apoptosis \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, it is unknown how M3AchR regulate p53-induced myocardial apoptosis.\u003c/p\u003e \u003cp\u003eAstragali Radix has been widely used as a functional food to protect cardiovascular, liver, and kidney diseases and improve health in traditional China or other East Asian countries. According to the U.S. Dietary Supplement Health and Education Act (DSHEA), it is categorized as a dietary supplement and consumed as an edible vegetable\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Astragali Radix has demonstrated protective properties against hyperlipidemia and myocardium offers benefits such as anti-inflammatory, antioxidation and lipid metabolism regulation\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Astragaloside IV (AS-IV), a triterpenoid saponin found in Astragali Radix, has significant potential as a dietary component for disease prevention\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In recent years, AS-IV has been consistently validated by numerous studies for its favorable myocardial protection effects\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. It has been reported that administering AS-IV (25ng/ml/d) could effectively protect rat myocardial cells from hypoxia and apoptosis through the MAPK and Erk2 pathways\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Moreover, AS-IV has the ability to delay the progression of myocardial fibrosis, cardiac hypertrophy, and heart failure\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Research suggests that AS-IV exerts its protective effects on myocardial inflammation and oxidative stress by regulating TGF-β1 and Nrf-2\u003csup\u003e34\u0026ndash;36\u003c/sup\u003e. Accordingly, AS-IV may protect myocardium from M3AchR inhibitors induced cardiomyocytes ischemia and apoptosis; however, the basic mechanisms remain unclear.\u003c/p\u003e \u003cp\u003eWe hypothesized that AS-IV could protect against myocardial apoptosis in a MI model by regulating the p53/Akt signaling pathway through M3AchR. This may provide a theoretical and experimental basis for elucidating the protective effect of AS-IV on MI through M3AchR receptors.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animal model and treatment\u003c/h2\u003e \u003cp\u003eAdult male C57BL/6 male mice (age: 6\u0026ndash;8 weeks) were purchased from Hangzhou Ziyuan Experimental Animal Technology Co. Ltd. (Hangzhou, China, Laboratory Animal Production). The experimental mice were raised under standard temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and humidity (40\u0026ndash;60%) and supplied with food, water, and adaptive feeding for 1 week.\u003c/p\u003e \u003cp\u003eAccording to the literature \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and the preliminary experimental results, the concentration of AS-IV (AS-IV) and the concentration of 4-DAMP and Ach \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e were selected, and all mice were randomly assigned to 6 groups (n\u0026thinsp;=\u0026thinsp;6 for each group): (1) control group; (2) MI group; (3) MI\u0026thinsp;+\u0026thinsp;4-DAMP (0.4\u0026micro;g/kg/d, ip) group; (4) MI\u0026thinsp;+\u0026thinsp;Ach (5mg/kg/d, ip) group; (5) MI\u0026thinsp;+\u0026thinsp;4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV (112mg/kg/d, ig) group; (6) MI\u0026thinsp;+\u0026thinsp;AS-IV group were selected in accordance with previous literature \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Pre-dose for 2 weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 MI mice model establishment\u003c/h2\u003e \u003cp\u003eThe mice were anesthetized with 1% pentobarbital (40 mg/kg) injected during left thoracotomy. The left anterior descending (LAD) coronary artery was ligated with a 7\u0026ndash;0 polypropylene suture, followed by skin suturing. Electrocardiogram (ECG) monitoring verified the success of MI surgery. T-wave changes and the ST-segment elevation in the ECG record confirmed that the mouse MI model was successfully established. All experimental protocols were approved by the Animal Experimentation Ethics Committee of the Anhui University of Chinese Medicine (AHUCM-mouse-2022009). All experiments were made to minimize animal suffering and conducted in accordance with guidelines and regulations. Our animal experimental research followed the \u003cem\u003ein vivo\u003c/em\u003e experimental (ARRIVE) guideline \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Before harvesting the tissue, animals were anesthetized with pentobarbital sodium (40 mg/kg) injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Electrocardiography measurement\u003c/h2\u003e \u003cp\u003eThe electrocardiogram of mice in each group was monitored one day after operation. After the mice were anesthetized with 1% pentobarbital (50mg/kg), the mice were placed on the mouse plate for fixation, and then the electrode needle was inserted into the corresponding position of the limbs in the way of the second lead to observe the electrocardiogram, and the changes in the heart rate of the electrocardiogram and the elevation level of the ST segment arch back were recorded. Each mouse was recorded three times for 120s each time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Echocardiography measurement\u003c/h2\u003e \u003cp\u003eThe small animal ultrasound imaging system (VINNO6LAB, Suzhou VINNO Technology Co., Ltd.) equipped with 18 MHz scanning connector was used to measure the echocardiography of mice. During the measurement, the mice were anesthetized with 1% pentobarbital (50mg/kg). Echocardiographic measurements were performed on left ventricular (LV) parasternal long-axis M-mode imaging. The mean value of the data is based on the measurement results of at least six cardiac cycles, including left ventricular end-systolic diameter (LVID), left ventricular end-diastolic diameter (LVIDd), and heart rate (HR). Left ventricular ejection fraction (EF) and left ventricular short axis shortening rate (FS) are calculated by VINNO6 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 TTC staining for infarct area measurement\u003c/h2\u003e \u003cp\u003eThe heart was carefully removed and rinsed with PBS buffer, and stored at -80\u0026deg;C for 20 min until frozen. Heart was then cutting into 5 equal thickness sections. The slices were placed in 1% 2,3,5-Triphenyltetrazolium Chloride (TTC) solution in a 37\u0026deg;C water bath for 15 min, followed by steeping in 10% neutralized formalin overnight. The heart slices were finally visualized using a Canon digital camera. The infarct area in the heart slices was presented in white, were calculated using Image J (1.53e) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Haematoxylin and eosin (HE) and Masson staining\u003c/h2\u003e \u003cp\u003eThe hearts were fixed in 4% paraformaldehydefor 24 h. Next, ethanol at varying concentrations was employed for dehydration, and xylene was utilized for infiltration. Then, 2-mm ventricle tissue slices were embedded with paraffin and stained with hematoxylin and eosin (H\u0026amp;E) and Masson. The histopathological changes were observed under an optical microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Tunel assay for myocardial apoptosis detection\u003c/h2\u003e \u003cp\u003eTunel staining with 3,3-diaminobenzidine (DAB) was used to detect myocardial apoptosis in heart slices. According to the instructions provided with the kit, heart slices were treated with the Tunel detection solution (5 uL of TdT enzyme and 45 uL of Biotin-dUTP) and incubated at 37\u0026deg;C for an hour. Next, the slices were treated with 50 uL of the streptavidin\u0026ndash;HRP working solution at room temperature for 30 minutes, then incubated with DAB and hematoxylin for 10 minutes at room temperature. The detection of cardiomyocyte apoptosis was performed with a Nikon eclipse 50i microscope. The normal cells appeared blue, while the Tunel-positive cardiomyocytes were brown.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Hoechst for myocardial apoptosis detection\u003c/h2\u003e \u003cp\u003eThe Hoechst 33342/PI dual staining kit from Biosharp (BL116A) was utilized for Hoechst staining experiments. For fixed tissue samples, the fixative was removed following fixation, and the samples were washed 2\u0026ndash;3 times with PBS buffer, each for 3\u0026ndash;5 minutes. Subsequently, an appropriate volume of 5 \u0026micro;L Hoechst 33342 staining solution was directly applied to cover the samples, followed by the addition of 5 \u0026micro;L PI staining solution. Incubation was carried out at 4\u0026deg;C in the absence of light for 20 minutes. After removing the staining solutions, the samples were washed twice with PBS buffer, each for 3\u0026ndash;5 minutes. Following mounting, the samples were observed directly under a fluorescence microscope. Hoechst 33342 utilizes ultraviolet light emitted by a krypton laser, with an excitation wavelength of 352 nm and an emission wavelength ranging from 400 to 500 nm, producing blue fluorescence. PI employs fluorescence induced by an argon ion laser, with an excitation wavelength of 488 nm and an emission wavelength exceeding 630 nm, generating red fluorescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Immunofluorescence study\u003c/h2\u003e \u003cp\u003eSlices of heart tissue were subjected to microwave antigen repair, blocked, and then incubated with a commercial primary anti-M3AchR (cat. no. AMR-006; 1:200; Thermo), anti-Akt (cat. no. AF6261, 1:200; Affinity Biosciences), anti-phosphorylated (anti-p)-Akt (cat. no. AF0016; 1:200; Affinity Biosciences) for 4\u0026deg;C overnight. After washing the heart slices three times with 0.01 M TBS (pH 7.4) for 5 minutes each, they were incubated at 37\u0026deg;C with TRITC-conjugated goat anti-rabbit IgG (cat. no. 139931; ZSGB-BIO, Beijing China) for 50 minutes. The nuclei were stained using a 4\u0026prime;,6-diamidino-2-phenylindole (DAPI; cat. no. G1012; Servicebio) solution in darkness for 5 minutes. The slides were finally imaged with a NIKON ECLIPSE C1 microscope, and the immunofluorescence was examined using Image J (1.53e) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Western blotting\u003c/h2\u003e \u003cp\u003eThe total proteins derived from C57 mouse hearts underwent fractionation with the RIPA cell lysate (containing 1-mm phenylmethylsulfonyl fluoride [PMSF]) and added to 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). Then, the proteins were transferred into the polyvinylidene fluoride (PVDF) membrane. The membranes were incubated with primary anti-Bcl-2 (1:1,000; cat. no. ab32124; Abcam), anti-Bax (cat. no. ab32503; 1:1,000; Abcam), anti-caspase-3 (cat. no. ab32351; 1:5,000; Abcam), anti-cleaved-caspase-3 (cat. no. 341034; 1:1,000; ZENBIO), anti-Akt (cat. no. AF6261; 1:1,000; Affinity Biosciences), anti-phosphorylated (anti-p)-Akt (cat. no. AF0016; 1:1,000; Affinity Biosciences), anti-hypoxiainducible factor (HIF)-1α (cat. no. AF1009; 1:500; Affinity Biosciences), anti-M3AchR (cat. no. PA5-85322; 1: 1,000; Thermo), anti-p53 (cat. no. AF0879; 1:1,000; Affinity Biosciences), anti-phosphorylated (anti-p)-p53 (cat. no. R380837; 1:500; ZENBIO), and β-actin (Zs-BIO) at 4\u0026deg;C overnight. Subsequently, the blotted membranes underwent 2 h of incubation with the horseradish peroxidase (HRP)-conjugated secondary antibodies at ambient temperature. Enhanced chemiluminescence (ECL) substrates (Millipore) contributed to the visualization of the signals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Detection of CK-MB, cTnI and LDH in serum\u003c/h2\u003e \u003cp\u003eSerum levels of CK-MB (cat. no. RX201007M; Quanzhou Ruixin Biological Technology Co., Ltd.; Quanzhou; China), cTnI (cat. no. H149-2-2; Nanjing Jiancheng Bioengineering Institute; Nanjing; China), and LDH (cat. no. A020-2-2; Nanjing Jiancheng Bioengineering Institute; Nanjing; China) were measured by experimental kits with a RT-6100 microplate analyzer (Rayto Life and Analytical Sciences Co.,Ltd.) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was performed for comparisons between the two groups. One-way ANOVA analysis was performed for \u0026gt;\u0026thinsp;2 groups with a post-hoc least significant difference (LSD) test to evaluate the significant differences between each group. Statistical data were analyzed with the SPSS 22.0 software. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of AS-IV on cardiac function of MI model through M3 receptor\u003c/h2\u003e \u003cp\u003eWe used parasternal left ventricular long-axis imaging to evaluate the cardiac function of mice in each group, and recorded the changes of left ventricular ejection fraction (EF), left ventricular short-axis shortening rate (FS), left ventricular end-diastolic diameter (LVIDd) and left ventricular systolic diameter (LVIDs), and compared the functional differences between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results showed that compared with the MI model group, the 4-DAMP group significantly reduced the left ventricular ejection fraction (EF) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB(a)) and short-axis shortening rate (FS) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB(b)) of mice, increased the left ventricular end-diastolic diameter (LVIDd) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB(c)) and systolic diameter (LVIDs) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB(d)) of mice with myocardial infarction, while the AS-IV group significantly weakened the above-mentioned effects of the 4-DAMP group. Compared with 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV group, AS-IV group significantly increased the left ventricular ejection fraction (EF) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB(a)) and short-axis shortening rate (FS) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB(b)) of mice, and decreased the left ventricular end-diastolic diameter (LVIDd) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB(c)) and systolic diameter (LVIDs) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB(d)) of mice with myocardial infarction, the difference was statistically significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 AS-IV exerts a protective effect on myocardial injury by activating M3AchR in mice myocardial ischemia model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBy observing the ST segment and T wave (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), we could find that compared with the control group, the ST segment and T wave of MI group and MI\u0026thinsp;+\u0026thinsp;4-DAMP group were significantly elevated, indicating that the heart had ischemia; MI\u0026thinsp;+\u0026thinsp;Ach and MI\u0026thinsp;+\u0026thinsp;AS-IV groups could significantly reverse the pathological state of ST segment and T wave in model mice. Compared with the MI\u0026thinsp;+\u0026thinsp;4-DAMP group, the ST segment and T wave of the MI\u0026thinsp;+\u0026thinsp;4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV group decreased slightly. The results showed that AS-IV could alleviate the abnormal elevation of ST segment in mice with myocardial ischemia induced by M3 receptor. The results of ECG heart rate showed that the heart rate of MI group was significantly lower than that of Control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC); Compared with MI group, the heart rate of 4-DAMP group was significantly higher, that of Ach group was significantly lower, and that of AS-IV group was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC); Compared with 4-DAMP group, the heart rate of 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV group was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC); Compared with 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV group, the heart rate of AS-IV group was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the assessment of myocardial infarct size (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), we observed that the 4-DAMP group significantly increased the myocardial infarct size in mice compared to the MI model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, after administration of AS-IV or the M3 receptor agonist ACh, the myocardial infarct size significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Compared to the 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV group, the AS-IV group significantly reduced the myocardial infarct size in mice, with a statistically significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Effect of AS-IV on myocardial injury indicators and pathological changes in a myocardial ischemia model via the M3 receptor\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis study aimed to explore the anti-M3AchR effect on myocardial injury \u003cem\u003ein vivo\u003c/em\u003e. After a week of pretreatment with 4-DAMP and Ach, the MI model for drug administration in each group was formed. When compared to the MI group, 4-DAMP exacerbated MI morphological changes including myocardial myolysis and inflammatory cells infiltration, and myocardial apoptosis staining with brownish yellow nuclei, while 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV improved 4-DAMP morphological changes and myocardial apoptosis on histological H\u0026amp;E staining assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Masson's trichrome staining was performed to detect fibrosis deposition in myocardium. Fibrous tissue stained blue and myocardial fibers stained red. Collagen content and disarray of myocardial fibers was significantly increased in the 4-DAMP group compared with the MI group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Compared with the 4-DAMP group, the collagen content and myocardial fiber disorder in the 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV group were significantly reduced. The LDH activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), CK-MB (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and cTnI (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) concentrations in 4-DAMP group plasma were significantly elevated than the MI group, while the 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV group lactate dehydrogenase (LDH) activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), CK-MB (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and cTnI (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) concentrations were decreased compared to the 4-DAMP group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of AS-IV on myocardial apoptosis in MI model via M3 receptor\u003c/h2\u003e \u003cp\u003eThe Tunel (DAB) and Hochest staining results indicate that, compared to the MI myocardial infarction model group, administration of the M3 receptor inhibitor 4-DAMP (0.4 mg/kg) significantly increased cardiomyocyte apoptosis in the myocardial tissue of ischemic mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B), suggesting that inhibiting the M3 receptor could enhance cardiomyocyte apoptosis. Compared to the MI myocardial infarction model group, the AS-IV group reduced cardiomyocyte apoptosis in ischemic mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B). Compared to the 4-DAMP group, the combination of 4-DAMP and AS-IV attenuated the inhibitory effect of AS-IV on cardiomyocyte apoptosis in ischemic mice, indicating that AS-IV affects cardiomyocyte apoptosis in ischemic mice by acting on the M3 receptor (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 AS-IV could significantly affect the expression of M3AChR in MI mouse model\u003c/h2\u003e \u003cp\u003eWestern-Blot and immunofluorescence staining results showed that administration of M3 receptor inhibitor 4-DAMP (0.4 mg/kg) significantly reduced the expression of M3AChR in myocardial tissues of mice with myocardial ischemia compared with the MI model group, suggesting that inhibition of M3AChR could reduce the expression of M3AChR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B). Compared with the MI model group, the AS-IV group significantly elevated the expression of M3 receptors in the myocardial tissues of mice with myocardial ischemia (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B). Compared with the 4-DAMP group, the combination of 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV attenuated the effect of 4-DAMP on the decrease of M3AChR expression in the myocardial tissues of mice with myocardial ischemia (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA,B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Analysis of the Binding Targets of AS-IV with M3AChR Protein\u003c/h2\u003e \u003cp\u003eAs depicted in the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the analysis conducted using the CB-Dock software indicates that there exist two binding pockets (Pocket1 and Pocket2) between AS-IV, serving as the ligand, and the M3AChR protein. The FitDock Score for these two pockets is -3.5 and 29, respectively, with the templates being t2:4j4q and t5:7bu6 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, there are five structural molecular binding sites (Pocket 3\u0026ndash;7) between AS-IV, acting as the ligand, and the M3AChR protein. The Vina Score for these sites is -9.1, -8.7, -7.8, -7.4, and \u0026minus;\u0026thinsp;7.3, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The results of the analysis predict that AS-IV, acting as a ligand, exhibits favorable binding characteristics with the M3AChR protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTemplete based docking results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePockets\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFitDock Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTemplate ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eTemplate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003et2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4j4q\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003et5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7bu6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStructure based docking results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePockets\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVina Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCavity Size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eCenter\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eX\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eZ\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1974\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1814\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.7 Identification of hub proteins and signaling pathways for the treatment of MI by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shown, we obtained gene targets related to myocardial hypoxia from GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/,v5.3.0\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/,v5.3.0\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e); Online Mendelian Inheritance in Man (OMIM, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.omim.org/\u003c/span\u003e\u003cspan address=\"http://www.omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, Updated June 26, 2021); Therapeutic Target Database (TTD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://db.idrblab.net/ttd/\u003c/span\u003e\u003cspan address=\"http://db.idrblab.net/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, Updated June 1, 2020), and Drugbank database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://go.drugbank.com/\u003c/span\u003e\u003cspan address=\"https://go.drugbank.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, version 5.1. 8). After the results of KEGG pathway top 20 pathway ranking were analyzed, it was concluded that myocardial hypoxia is closely related to HIF-1α and apoptosis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). From the analysis of the results of p53 pathway map in KEGG, it could be concluded that under hypoxic myocardium, the differentially expressed genes were mainly enriched in the pathways related to Bcl-2, Caspase-3 and PAI. And the results of gene enrichment pathway related to Bcl-2 and Caspase-3 pointed to apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.8 AS-IV regulated the expression of HIF-1α through M3AChR under MI model\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the administration of the M3AChR inhibitor 4-DAMP (0.4 mg/kg) was able to significantly increase the expression of HIF-1α in myocardial tissues of mice with myocardial ischemia compared with the MI infarction model group, suggesting that inhibition of the M3AChR was able to increase the phenomenon of posthypoxia in myocardial ischemia (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Compared with the MI infarction model group, the AS-IV group significantly decreased the expression of HIF-1α in the myocardial tissues of myocardial ischemic mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Compared with the 4-DAMP group, the combination of 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV attenuated the increase of HIF-1α expression in the myocardial tissues of myocardial ischemic mice by 4-DAMP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.9 AS-IV could act on M3AChR to reduce the expression of p-p53/p53 and p-Akt/Akt and thereby improving MI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWestern-Blot and immunofluorescence results showed that 4-DAMP (0.4 mg/kg), an M3AChR inhibitor, significantly increased the expression of p-p53/p53 and decreased the expression of p-Akt/Akt in myocardial tissues of mice with myocardial ischemia compared with the MI infarction model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e), suggesting that the inhibition of M3AChR could increase the expression of p-p53/p53 and decreased the expression of p-Akt/Akt. Compared with the MI infarction model group, the AS-IV group significantly reduced the expression of p-p53/p53 and increased the expression of p-Akt/Akt in the myocardial tissues of mice with myocardial ischemia (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Compared with the 4-DAMP group, the combination of 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV attenuated the reduction of p-p53/p53 and increase of p-Akt/Akt expression in the myocardial tissues of mice with myocardial ischemia (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.10 Effect of AS-IV on apoptosis rate and apoptotic proteins Bax, Bcl-2 and Caspase-3 in MI model via M3AChR\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWestern-Blot results showed that compared with the MI group, administration of the M3AChR inhibitor 4-DAMP (10 \u0026micro;M) was able to significantly increase the protein expression of Bax, Caspase-3 and cleaved-Caspase-3 and decrease the protein expression of Bcl-2 in hypoxic myocardium (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). It is suggested that inhibition of M3AChR could play a pro-apoptotic role by activating the protein expression of Bax, Caspase-3 and cleaved-Caspase-3 and inhibiting the protein expression of Bcl-2. Compared with the MI group, the AS-IV group decreased the expression of Bax, Caspase-3 and cleaved-Caspase-3 and elevated the expression of Bcl-2 in hypoxic myocardium (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Compared with the 4-DAMP group, the combination of 4-DAMP\u0026thinsp;+\u0026thinsp;AS-IV attenuated the decreasing effect of AS-IV on the expression of Bax, Caspase-3 and cleaved-Caspase-3 and the elevating effect on the expression of Bcl-2 in hypoxic myocardium (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn recent years, the regulation of cardiomyocytes function by the M3AchR has garnered widespread attention within the cardiovascular research community\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Studies have shown that the M3AchR inhibitor 4-DAMP could significantly increase the effects of myocardial injury and apoptosis after hypoxia\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, while the M3AchR agonist Ach could protect against myocardial injury and apoptosis after ischemia\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. AS-IV, as the primary active ingredient in Astragali Radix, exhibits well-defined myocardial protection after ischemia. Therefore, this study first investigates whether AS-IV protects the symptoms of MI through M3AchR. In this research, we replicated a mouse MI model by ligating the left anterior descending branch (LAD), and observed changes in the mouse heart through electrocardiogram, TTC staining, serum biomarkers and pathological experiments. To investigate whether AS-IV affects cardiac function and myocardial injury after MI through the M3AchR, we used specific M3AchR inhibitors 4-DAMP and agonists Ach to observe and study the effects of AS-IV on the regulation of M3AchR in MI mice. The results indicated that administration of M3AchR inhibitor 4-DAMP could mitigate cardiac function in mice with MI, exacerbate myocardial injury and fibrosis after ischemia; AS-IV could protect against ischemia induced myocardial injury and fibrosis exacerbation caused by 4-DAMP, and improve cardiac function in the body \u003cem\u003ein vivo\u003c/em\u003e. This study suggests that AS-IV improves cardiac function in MI mice and has a protective effect on myocardial injury and fibrosis after ischemia, which is associated with agonizing the M3AchR. Thus, AS-IV could be a beneficial dietary option or combination to ease MI symptoms through M3AchR, potentially broadening its application scope in everyday health practices.\u003c/p\u003e \u003cp\u003eNotably, it is necessary to understand how AS-IV acts on M3AchR to exert MI protection. This experiment first demonstrated that AS-IV could improve the downregulation of M3 receptor expression by ischemia. This aligns with earlier findings that enhancing M3AchR expression might avert cardiac death due to MI\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Moreover, our analysis further revealed seven highly probable drug targets for AS-IV to interact with the M3AchR. To delve deeper into the mechanism of AS-IV's myocardial protective action, we employed CB-Dock (Cavity-detection Guided Blind Docking) to analyze the low-molecular-weight protein interactions between AS-IV and the M3AchR. Through CB-Dock analysis, we identified potential molecular targets for AS-IV with the M3AchR. This suggests that AS-IV may exert its MI protective effects by directly act on M3AchR.\u003c/p\u003e \u003cp\u003eThe M3AchR inhibitor effect promoting MI is supposed to correlate with apoptosis in cardiomyocytes. However, the mechanism of myocardial apoptosis caused by M3AchR inhibitor induced MI injury has not been elucidated yet. Therefore, exploring the protective mechanism of M3AchR against MI provides a preliminary basis for demonstrating the mechanistic impact of AS-IV on M3AchR. The pathway triggered by MI that leads to cell apoptosis could be mediated by central mediator changes to Bax/Bcl-2 and caspase-3 activation, which is regulated by the p53 protein \u003csup\u003e\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, as well as the involvement of HIF-1α upregulation \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Through KEGG pathway analysis, we found a strong correlation between myocardial ischemic injury and the apoptotic pathway. Our research results indicate that inhibiting M3AchR increases the expression of HIF-1α, Bax, and cleaved Caspase-3/Caspase-3, while reducing the expression of Bcl-2, exacerbating the apoptotic changes of ischemic myocardium. Phosphorylated Akt, activated by phosphatidylinositol-3 kinase, is a vital mediator in cell survival and the anti-apoptotic process\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Moreover, p53 activation could alleviate the Akt protein expression level in the mutant p53 mouse model \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e and implicated in the cardiomyocytes proliferation and apoptosis \u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The results showed that M3AchR inhibitor-treated ischemic myocardium promoted p-p53/p53 expression and repressed p-Akt/Akt expression. Cumulatively, inhibition of M3AchR could exacerbate apoptosis in ischemic myocardium via regulating p53 expression and Akt phosphorylation.\u003c/p\u003e \u003cp\u003eFinally, we investigated the regulation of apoptosis and p53/Akt related apoptotic protein expression in mice with MI by AS-IV through M3AchR. The results show that inhibiting the M3AchR increases the expression of HIF-1α protein, while AS-IV decreases its expression through the M3AchR, suggesting a significant correlation between the regulation of M3AchR and AS-IV's protective effects with the degree of MI. By employing M3AchR inhibitors, we found that AS-IV reduced myocardial apoptosis in ischemic mice through M3AchR. Subsequently, Western-Blot results indicate that AS-IV increases Bax and cleaved-Caspase-3/Caspase-3 expression and decreases Bcl-2 expression in MI through the M3AchR. We measured p-p53/p53 and the downstream p-Akt/Akt levels to elucidate how the AS-IV regulates p53- and Akt-induced apoptosis in cardiomyocytes under MI through M3AchR. Inhibiting the M3AchR enhances p-p53/p53 protein expression, while AS-IV decreases its expression through the M3AchR. Moreover, inhibiting M3AchR could reduce the expression of p-Akt/Akt in myocardium, while AS-IV increases p-Akt/Akt through M3AchR. The findings of this study indicate that p53 and Akt is an important link in AS-IV's mechanism for inhibiting myocardial apoptosis. Therefore, AS-IV could act on M3AchR to alleviate MI symptoms, improve heart function and myocardial injury in mice, and inhibit myocardial apoptosis in ischemic mice through the p53/Akt pathway.\u003c/p\u003e \u003cp\u003eIn summary, this study is the first to apply the M3AchR to investigate the mechanism of AS-IV in inhibiting myocardial apoptosis under MI model. By using morphological, bioinformatics, and molecular biology methods, our research revealed that AS-IV could attenuate the ischemic injury and apoptosis aggravation of cardiomyocytes by M3AchR inhibitors in vivo. Moreover, the anti-apoptotic effect of AS-IV might relate to the regulation of p53/Akt signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). However, further research is needed to verify whether AS-IV could directly interact with M3AchR or have other forms of impact. This study provides new experimental evidence to reveal the cardioprotective effect of AS-IV through M3AchR and p53/Akt signaling pathway. Crucially, this research sets a foundation for the application of AS-IV as a nutritional adjunct to relieve MI symptoms.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eASSOCIATED CONTENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData Availability Statement Available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYixuan Lin\u003c/strong\u003e - Department of Endocrinology, The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, Anhui, 230031, China; Phone: +8615256595986; Email: [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQiang Zuo\u003c/strong\u003e - Department of Cardiology, The First Affiliated Hospital, Anhui University of Chinese Medicine, Hefei, Anhui, 230031, China; Phone: +8613605691870; Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCS, QZ and YL designed the research and revised the manuscript. CS, SC, FW, LS, LY and QZ conducted the experiments, and involved in the data acquisition, and manuscript writing. CS, FW and QZ analyzed and interpreted the data. All authors have read and approved the final manuscript. YL and QZ confirm the authenticity of all the raw data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (no. 81904147), the Youth Research Project of Health Commission of Anhui Province (grant no. AHWJ2023A30241), the Scientific Research Foundation of Education Department of Anhui Province of China (grant no. 2024AH051007), the Scientific Research Foundation of Education Department of Anhui Province of China (grant no. 2023AH050867).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSassu, E.; Tumlinson, G.; Stefanovska, D.; Fernandez, M. C.; Iaconianni, P.; Madl, J.; Brennan, T. A.; Koch, M.; Cameron, B. A.; Preissl, S.; Ravens, U.; Schneider-Warme, F.; Kohl, P.; Zgierski-Johnston, C. 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However, it is unclear whether AS-IV could protect myocardium from ischemia by activating M3 subtype of muscarinic acetylcholine receptor (M3AchR) receptors and regulate cardiomyocytes apoptosis through p53/Akt. This study aims to establish an in vivo model of myocardial ischemia (MI) and utilize morphological, bioinformatics, and molecular biology methods to elucidate the mechanism by which AS-IV regulates MI and apoptosis through the M3AchR and p53/Akt pathway. The results suggested that AS-IV was able to alleviate the MI injury and aggravated apoptosis of cardiomyocytes caused by M3AchR inhibitors 4-DAMP in vivo. Moreover, AS-IV may have a protective effect on MI by directly acting on M3AchR. Mechanistically, AS-IV's anti-apoptotic effect could be associated with the regulation of the p53/Akt signaling pathway. Collectively, our research indicates that AS-IV could alleviate MI and exert myocardial protective effects by acting on the M3AchR and p53/Akt signaling pathways. This study provides a theoretical basis for exploring potential protective targets of AS-IV and elucidating new functions and mechanisms of AS-IV.\u003c/p\u003e","manuscriptTitle":"AS-IV alleviates blocking of M3AchR induced myocardial apoptosis through p53/Akt signaling pathway under myocardial ischemia model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-30 09:50:54","doi":"10.21203/rs.3.rs-6667733/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43fe0c80-f74b-44fd-be83-b2a09a9fe8c0","owner":[],"postedDate":"June 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50734480,"name":"Health sciences/Health care/Nutrition"},{"id":50734481,"name":"Health sciences/Cardiology"},{"id":50734482,"name":"Health sciences/Cardiology/Cardiovascular biology"},{"id":50734483,"name":"Health sciences/Diseases"},{"id":50734484,"name":"Health sciences/Diseases/Cardiovascular diseases"},{"id":50734485,"name":"Biological sciences/Drug discovery/Pharmacology/Receptor pharmacology"},{"id":50734486,"name":"Biological sciences/Drug discovery/Drug regulation"},{"id":50734487,"name":"Biological sciences/Chemical biology/Mechanism of action"},{"id":50734488,"name":"Biological sciences/Chemical biology/Pharmacology/Receptor pharmacology"}],"tags":[],"updatedAt":"2025-07-14T18:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-30 09:50:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6667733","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6667733","identity":"rs-6667733","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}