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Huoxin pill prevents acute myocardial ischaemia injury via inhibition of Wnt/β‑catenin signaling

Abstract Myocardial infarction (MI) is one of the leading causes of death worldwide, and due to the widespread and irreversible damage caused, new therapeutic treatments are urgently needed in order to limit the degree of ischaemic damage following MI. Aberrant activation of Wnt/β‐catenin signalling pathway often occurs during cardiovascular diseases including MI, which results in excess production of reactive oxygen species (ROS) and further promotes myocardial dysfunction. Huoxin pill (HXP) is a Traditional Chinese Medicine formula that has been widely used in the treatment of coronary heart disease and angina; however, its mechanisms remain unclear. Here, we performed mouse models of MI and examined the effects and mechanisms of HXP in protecting against MI‐induced ischaemic damage. Our study showed that administration with HXP robustly protected against MI‐induced cardiac injuries, decreased infarct size and improved cardiac function. Moreover, HXP attenuated ischaemia‐induced DNA damage occurrence in vivo and H2O2‐induced DNA damage occurrence in vitro, via potent inhibition of adverse Wnt/β‑catenin signalling activation. Our study thus elucidated the role and mechanism of HXP in protecting against MI and oxidative stress‐induced injuries and suggests new therapeutic strategies in ischaemic heart disease via inhibition of Wnt/β‐catenin signalling pathway.

Interestingly, adverse activation of Wnt/β-catenin signalling following ischaemia is strongly linked with the enhanced production of reactive oxygen species (ROS) that results in DNA damage occurrence and serves as an important mediator of various pathophysiological signals involved in myocardial cell death. [19][20][21] Excessive production of ROS promotes myocardial dysfunction and is evident in patients with acute MI and chronic heart failure. 22 Indeed, oxidative stress-induced DNA damage occurrence has been proposed as a key mechanism of cardiomyocyte death during cardiac ischaemia. 23 Therefore, the inhibition of ROS via antioxidant drugs or agents that protected against oxidative stress is critical in preventing cardiac damage following MI.
Huoxin Pill (HXP) is a Traditional Chinese medicine (TCM) formula that is composed of the following ingredients: Panax ginseng, Ganoderma lucidum, Moschus, Aconiti Lateralis Radix Praeparata, Carthami Flos, Bovis Calculus Sativus, Margarita, bear bile, Bufonis Venenum and Borneolum Syntheticum and has been widely used as an alternative and complementary medicine for the treatment of coronary heart disease and angina pectoris throughout Asia. 24 Numerous components in HXP, Ginsenosides from Panax ginseng, Ganoderic acids from Ganoderma lucidum, and flavonoids including Hydroxysafflor Yellow A from Carthamus tinctorius L. have been shown to exhibit antioxidative effects that may protect against cardiac injury [25][26][27] ; however, the underlying mechanisms of HXP in the treatment of myocardial infarction remains largely unknown.
Our current study aimed to examine the cardioprotective effects of HXP in preventing ischaemic damage and improving cardiac function following myocardial infarction, as well as elucidating the function of HXP in ameliorating oxidative stress-induced DNA damage occurrence via inhibition of Wnt/β-catenin signalling pathway.

| Preparation of HXP extract
To prepare the extract, HXP was grinded or pounded into a fine powder that passes through a sieve with nominal mesh aperture of 180 µm. The dried powder was re-dissolved in PBS to a concentration of 10 mg ml −1 , filtered with a 0.22 µm filter and stored at −20 °C for further use. For in vivo studies, HXP were used at final concentrations of 3 mg kg −1 (low-dose group) or 9 mg kg −1 (high-dose group). For in vitro studies, HXP was used at the indicated final concentrations.
HXP and PBS were administered via oral gavage every second day from one week prior to MI operation. Subsequently, MI was performed as previously described. 21 Briefly, mouse was anaesthetized, and ligation of the proximal left anterior descending coronary artery was performed in all groups other than sham group that was subjected to the same procedure but without ligation. Mice were sacrificed via intraperitoneal injection of sodium pentobarbital (225 mg kg −1 ) euthanasia solution.

| Quantitative Real-time PCR
For quantitative real-time PCR analysis, RNA was extracted using TRIzol reagent (Takara, Japan) and reverse transcribed to cDNA using a Prime Script RT reagent Kit according to the

| Histological staining and Immunofluorescence
For histology analysis, hearts were fixed with 4% paraformaldehyde overnight, paraffin-embedded, and sectioned (6 µm). Heart sections were stained with Masson's trichrome staining for visualization of infarcted area under a light microscope (Leica, Germany). Infarct size (%) was determined according to the ratio of endocardial infarct circumference compared with total left ventricular endocardial circumference ×100. For immunofluorescence, hearts were fixed with optimal cutting temperature (OCT) compound and frozen prior to sectioning.
Slides were fixed in 4% paraformaldehyde, permeabilized with 0.25% Triton X-100 in PBST (0.5% Tween in PBS), blocked with 5% bovine serum albumin (BSA) and incubated with the respective primary antibodies overnight at 4 °C. Following washing and incubation with secondary antibody, nuclei were stained with DAPI, and slides were mounted prior to imaging using a Zeiss LSM 710 confocal microscope.

Adult ventricular cardiomyocytes AC16 cells were used for in vitro
experiments. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum, 100 U ml −1 penicillin and 100 mg ml −1 streptomycin, and cultured in a 37 ˚C humidified incubator supplemented with 5% CO 2 . AC16 cardiomyocytes were pretreated with HXP (1 µg ml −1 ) or PBS for 2 hours,

| Western blotting
Total protein was extracted with RIPA lysis buffer. Nuclear protein was extracted by nucleoprotein extraction kit (Sangon Biotech, China). Proteins were separated on 12% SDS-PAGE gels and then transferred onto a 0.22 µm PVDF membrane, blocked with 5% non-fat milk for 1 hour and incubated with the primary antibodies: antiβ-catenin, antiγ-H2AX, anti-TBP or GAPDH overnight at 4 ˚C.
Subsequently, membranes were incubated with the secondary antibodies for 2 hours at room temperature and resulting protein bands were detected via chemiluminescence.

| Statistical analysis
Statistical analysis was performed using SPSS 26.0 software (Chicago, IL, USA). Data were expressed as the mean ± standard error of mean (sem). Independent samples t test or Mann-Whitney U test was performed on data with normal distribution or skewed distribution, respectively. P values <0.05 were considered statistically significant.

| HXP improves cardiac function following ischaemic injury
In order to evaluate the cardioprotective effect of HXP following ischaemia, we performed mouse models of myocardial infarction (MI) in mice administered with either PBS or HXP at both low and high doses. Cardiac function was evaluated via echocardiography at 4 weeks following MI, which showed significant decreases in left ventricular (LV) ejection fraction (EF%), fractional shortening (FS%), as well as stroke volume (SV) values compared to SHAM-operated mice ( Figure 1A, B). Administration with HXP significantly improved cardiac function compared to PBS-treated mice, with even a low dose of HXP having more significant cardioprotective benefits. Moreover, M-mode echocardiography at the border of the infarct zone exhibited significant improvements in LV wall movement in HXP-administrated mice compared to PBS-administrated mice ( Figure 1C) with equivalent heart rates between groups ( Figure 1D). LV end-diastolic wall thickness in diastole (LVED;d) was decreased at the zone bordering the infarct area following MI compared to SHAM-operated mice ( Figure 1E), which was to some extent attenuated in HXP-treated mice.
Interestingly, there was an increase in LVED;d wall thickness at the remote zone of LV near the papillary muscles at 4 weeks post-MI, which was more pronounced in HXP-treated mice, suggesting a protective over-compensatory mechanism via increasing remote LV wall thickness post-MI ( Figure 1F). The summary of cardiac function parameters was shown in ( Figure 1G). These results indicate that HXP has a robust effect in improving cardiac function following myocardial ischaemic injury.

| HXP prevents MI-induced cardiac remodelling
We further examined the degree of cardioprotection following myocardial infarction by examining the infarct size at 4 weeks following MI. Masson's trichrome staining showed that mice administrated with HXP (both low-dose and high-dose) had significant reductions in infarct size in the ischaemic heart compared to PBS-administrated mice (Figure 2A, B). Notably, the infarct zones were generally reduced across all sectioning levels in HXP-administered mice compared to PBS-administered mice, indicative of a decreased degree Interestingly, a low dose of HXP already provided adequate cardioprotective benefits, while a higher dose further improved several parameters, demonstrating the robust effect of HXP in protecting the heart against ischaemic injury.

| HXP protects against ischaemia-induced DNA damage in vivo and in vitro
Reactive oxygen species (ROS) is widely known to play an important role in the development of cardiovascular diseases, in particular, during acute myocardial infarction. Unrepaired DNA damage and oxidative stress induced by ischaemia is the leading cause for cardiac cell death and is a common mechanism for acute or chronic heart diseases. 29 Thus, we investigated the role of HXP

| HXP prevents ischaemia-induced cardiac injury and DNA damage via potent inhibition of Wnt/β-catenin signalling
There is accumulating evidence that suggests Wnt/β-catenin signalling pathway plays a central role in ischaemia-induced cardiac injury. To further investigate the underlying mechanism by which HXP prevents ischaemic injury and oxidative stress-induced DNA damage, we examined the effect of HXP on the activation of Wnt/β-catenin pathway using β-catenin-responsive TOPflash gene assays. Wnt/β-catenin signalling was significantly activated upon stimulation with Wnt ligand Wnt3a, Wnt agonist LiCl and Wnt receptor LRP6 ( Figure 4A-C).
Surprisingly, HXP administration exhibited a potent and specific inhibitory effect on the activation of TOPFlash in a dose-dependent manner, demonstrating that HXP is a robust agent in inhibiting the activation of Wnt/β-catenin signalling pathway ( Figure 4A-C).
We next performed siRNA knockdown assays of β-catenin, which showed that following H 2 O 2 treatment, knockdown of F I G U R E 4 HXP is a potent inhibitor of Wnt/β-catenin activation-induced cardiac injury and DNA damage.  Figure 4A). Notably, HXP-treated mice reduced the levels of nuclear beta-catenin accumulation compared to PBS-administered mice following MI even in the remote region, although this effect was slightly less pronounced than in the infarcted region.
Notably, mice administered with HXP had markedly higher levels of cytoplasmic phosphoβ-catenin, but reduced levels of nuclear β-catenin accumulation compared to PBS-administered mice in a dose-dependent manner in both the infarct region ( Figure 4E, F) and the remote region (Supplemental Figure 4A

| DISCUSS ION
Acute myocardial infarction is the leading cause of death worldwide. 30 There is accumulating evidence that suggests Wnt signalling pathway is activated during the wound healing process following MI.

Studies have demonstrated that injections of recombinant Wnt3a
following MI led to a worsened cardiac function and increased infarct size. 31 Moreover, stabilization of cardiomyocyte β-catenin expression further deteriorated cellular function following cardiac insults. 17 In contrast, inhibition of Wnt pathway, including secreted Frizzled-related proteins, [32][33][34][35] small molecular inhibitors such as SEN195, pyrvinium, WNT974 or ICG001 have been shown to alleviate pathological remodelling and improve cardiac function following MI. 18,[36][37][38] Further, our previous study demonstrated that inhibition and cardiac-specific deletion of β-catenin protected the heart from ischemic injury. 17 Inhibition of Wnt signalling pathway should have therefore a general protective effect following myocardial infarction, and drugs that can minimize cardiac ischaemic injury via inhibiting adverse activation of Wnt/β-catenin signalling is urgently needed.
Numerous studies have demonstrated that increased reactive oxygen species (ROS) promotes myocardial dysfunction and cardiac damage, which can lead to heart failure. 22 Increased ROS has also been shown to exacerbate cardiomyocyte damage and apoptosis following MI. 39 Due to the fact that cardiac ischaemia triggers rapid release of ROS that subsequently leads to widespread DNA damage, the inhibition of ROS is crucial in attenuating cardiac damage following MI. Various antioxidant drugs have shown promising effects in protecting against MI-induced cardiac damage both in animal models and clinically. 40,41 Activation of Wnt/β-catenin signalling has also been shown to enhance ROS production, while its inhibition not only prevents ROS, but also had protective effects following MI. 17 Thus, the ability of HXP to not only inhibit Wnt/β-catenin signalling activation following MI, but also prevent ROS production, demonstrated its effectiveness in the treatment of cardiac ischaemia.
Due to the complex pathogenesis and limited effective treatment options for acute MI, there is still an urgent need for drugs that can minimize cardiac ischemic injury or improve cardiac function following MI. Traditional Chinese medicine (TCM) has been widely used for the treatment of various cardiovascular diseases in China and numerous Asian countries. One such TCM prescription is HXP, which has been clinically used in the treatment of coronary heart diseases. 24 Our current study demonstrated for the first time that HXP acts as a robust inhibitor of Wnt/β-catenin signalling, thereby strongly protecting against ischaemic damage and improving cardiac function following MI. Notably, HXP administration not only attenuated cardiac ischaemic damage and scar formation, it also resulted in drastic improvements in cardiac function. These findings provide important insights into the roles and mechanisms of HXP, and due to the ease of HXP drug application via oral intake implicate its use for the clinical treatment of myocardial infarction.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Qing Wang: Formal analysis (equal); Investigation (lead); Visualization

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.