Therapeutic effects of higenamine combined with [6]‐gingerol on chronic heart failure induced by doxorubicin via ameliorating mitochondrial function

Abstract Higenamine (HG) is a natural benzylisoquinoline alkaloid isolated from Aconitum with positive inotropic and chronotropic effects. This study aimed to investigate the possible cardioprotective effects of HG combined with [6]‐gingerol (HG/[6]‐GR) against DOX‐induced chronic heart failure (CHF) by comprehensive approaches. DOX‐induced cardiotoxicity model in rats and H9c2 cells was established. Therapeutic effects of HG/[6]‐GR on haemodynamics, serum indices and histopathology of cardiac tissue were analysed. Cell mitochondrial energy phenotype and cell mitochondrial fuel flex were measured by a Seahorse XFp analyser. Moreover, UHPLC‐Q‐TOF/MS was performed to explore the potential metabolites affecting the therapeutic effects and pathological process of CHF. To further investigate the potential mechanism of HG/[6]‐GR, mRNA and protein expression levels of RAAS and LKB1/AMPK/Sirt1‐related pathways were detected. The present data demonstrated that the therapeutic effects of HG/[6]‐GR combination on CHF were presented in ameliorating heart function, down‐regulation serum indices and alleviating histological damage of heart tissue. Besides, HG/[6]‐GR has an effect on increasing cell viability of H9c2 cells, ameliorating DOX‐induced mitochondrial dysfunction and elevating mitochondrial OCR and ECAR value. Metabolomics analyses showed that the therapeutic effect of HG/[6]‐GR combination is mainly associated with the regulation of fatty acid metabolites and energy metabolism pathways. Furthermore, HG/[6]‐GR has an effect on down‐regulating RAAS pathway‐related molecules and up‐regulating LKB1/AMPKα/Sirt1‐related pathway. The present work demonstrates that HG/[6]‐GR prevented DOX‐induced cardiotoxicity via the cardiotonic effect and promoting myocardial energy metabolism through the LKB1/AMPKα/Sirt1 signalling pathway, which promotes mitochondrial energy metabolism and protects against CHF.


| INTRODUC TI ON
Chronic heart failure (CHF) is the end stage of various cardiovascular diseases (CVDs). It is often accompanied by changes in the utilization of energy metabolic substrates. Also, abnormal mitochondrial function and dysfunction of energy metabolism of cardiac myocytes could lead to the progress of myocardial systolic dysfunction and left ventricular remodelling. 1 In the advanced stages of heart failure (HF), irreversible changes often occur due to the energy metabolism disorder of cardiac myocytes. 2 At this time, due to the reduction in mitochondrial biosynthesis and the change in mitochondrial function, mitochondrial energy metabolism is decreased resulting in the reduced ATP production, and cardiac myocytes are in the state of "energy starvation." Recent years, the relationship between HF and myocardial energy metabolism has become a hot topic in clinical research. Drugs which can enhance myocardial mitochondrial energy metabolism may have the potential effect to treat CHF. 3 Traditionally, the compatibility of Aconiti Lateralis Radix Praeparata (ALRP, Fuzi in Chinese) and Zingiberis Rhizoma (ZR, Ganjiang in Chinese) is a common pair of medicines in the treatment of HF, coronary heart disease (CAD), myocardial infarction and other CVDs. Studies have shown that ALRP has cardiotonic and anti-HF effects. The water-soluble components of ALRP, including higenamine (HG), salsolinol, coryneine chloride, fuzinoside and uracil, have been reported to be cardio cardiotonic tonic activities. 4 ZR has a protective effect on the hypoxic and hypoglycaemic injury of cardiac myocytes. It can inhibit platelet aggregation and improve ventricular systolic and diastolic function. Simultaneously, ZR can also reduce peripheral resistance, which has a protective effect on the formation of acute heart failure (AHF) model. 5 According to the theory of traditional Chinese medicine (TCM), both ALRP and ZR are pungent in taste and hot in nature. As a common clinical medicine pair from ancient times to nowadays, they are one of the most typical representatives reflecting the very essence of the theory of Chinese material medical compatibility. In the previous researches, the objective truth that "ALRP not exhibiting its hot property without ZR" has been proved in animals by the energy metabolism of mitochondria. 6 However, the active components and key mechanism regulating energy metabolism remain poorly understood.
Higenamine is a plant-based alkaloid initially isolated from the root of Aconitum and has been identified as the active cardiotonic component of Aconitum. As a traditional herb medicine, aconite has been used alone or in combination with other drugs for the treatment of HF in oriental Asia for millenaries. 7,8 Structurally, HG is similar to catecholamines, which can activate both β1-and β2-adrenergic receptors (AR). β2-AR activation explains the pharmacological effect of HG/aconite in treating CHF by enhancing the contractile response and reducing cardiomyocyte apoptosis. 9 Also, HG is identified as a novel α1-AR antagonist, which may contribute to its hypotensive effect and suppression platelet aggregation. 10 Pharmaceutically, it has multiple pharmacological effects, including positive inotropic effect, 11 dilations of blood vessels and bronchi, 12 anti-inflammatory activity, 13 antispasmodics, 14 anti-thrombotic, 15 anti-oxidative 16 and anti-apoptotic properties. 17 Relevant pharmacological effects and mechanisms have been reported in detail. Previous studies have shown that HG has great value on treating coronary artery disease (CVD), bradyarrhythmia, heart failure, arthritis, 11 cerebral ischaemia-reperfusion injury 18 and bronchoconstriction diseases. 19 Functional studies of isolated cardiomyocytes demonstrated its effectiveness in enhancing a contractile response. 9 Positive inotropic drugs have been used for many years to treat patients with AHF accompanied by reduced ejection. 20 Of note, based on the potential positive inotropic, dilating vascular and chronotropic effects, HG is an effective vasodilator in haemodynamics. It can reduce aortic impedance and systemic vascular resistance, thereby reducing afterload. These effects are similar to those of isoproterenol (ISO). Recent years, HG was approved by the China Food and Drug Administration for the clinical study. Currently, the phase III clinical trial of HG has been completed and achieved good results. 21 These properties suggest that HF could be potentially treated using HG with its inotropic and chronotropic effects. Studies specifically designed to assess the potential role and mechanism of HG in the treatment of HF are necessary.
Ginger has been widely used as traditional medicine for thousands of years. 6-gingerol ([6]-GR), a major bioactive constituent of ginger, is the most abundant and pungent gingerol in ginger. It has many effective pharmacological effects, such as cardiotonic, anti-angiogenesis, anti-inflammatory, anti-cancer, antipyretic and anti-ageing. 22 Notably, [6]-GR is a novel angiotensin II receptor (AT1) antagonist and offers some beneficial effects on cardiovascular diseases. 23    Rats were intraperitoneally injected with the corresponding drugs once a day for 7 days. HG and [6]-GR in 5 mg/kg/d showed superior efficacy in our previous study. Heart function was detected after the final injection. Serum samples and heart tissue were collected and stored at −80°C for the following detection.

| Detection of pharmacodynamic indicators
Serum levels of renin (RE), angiotensin II (Ang-II), aldosterone (ALD) and endothelin 1 (ET-1) were detected by a Synergy hybrid reader (BioTek). Left ventricular myocardial tissue was cut longitudinally and fixed with 4% paraformaldehyde. Haematoxylin-eosin (H&E) staining was performed for detecting myocardial morphological changes. Paraffin-embedded sections were observed under a Nikon microscope and by a Pro-Plus 7200 software.

| Cell culture and cell viability assay
The H9c2 rat cardiomyocyte cell lines were obtained from the Cell  sheath gas flow rate, 12 L/min; nozzle voltage, 500 V.

| Data processing and multivariate statistical analysis
Statistical analysis in MetaboAnalyst 4.0 26 online system was used to normalize the original data. The exported "data_normalized.

| Analysis of cell energy phenotype and cell extracellular flux
Seahorse XFp Cell Energy Phenotype Test and Extracellular Flux were analysed using the Seahorse XF test report analysis.  Table S1. For the data analysis, the relative mRNA expression levels of these genes were calculated based on 2 −ΔΔCt calculations to the β-actin endogenous reference.

| Immunohistochemical staining
Immunohistochemistry staining was performed as follows. The cardiac tissue was fixed with 4% paraformaldehyde and embed-

| Statistical analysis
Statistical analysis was performed using SPSS 23.0 software program (Chicago, United States) and GraphPad Prism 8.2.0 software (GraphPad Software). All data were presented as the mean ± standard deviation (SD). The differences between the two groups or multiple groups were calculated by a t test and one-way analysis of variance (ANOVA). The difference was considered statistically significant at P < .05 and highly significant at P < .01.

| Effects of HG/[6]-GR on cardiac function in rats
The results in Table S2 showed that LVSP and +dp/dt max decreased significantly in the DOX group, while LVEDP and −dp/dt max increased substantially in the DOX group. The +dp/dt max reduced to 50% of the control group indicated that the CHF rat model

| Serum levels of renin, Ang-II, ALD and ET-1 in various groups
As shown in Figure 1E-H, compared with the control group, serum levels of renin, Ang-II, ALD and ET-1 in the DOX group were markedly increased (P < 0.01). Compared with the DOX group, these indices were significantly reduced when rats were treated with DH and HG/[6]-GR (P < 0.01). Notably, these serum biochemical indicators had been significantly reduced by treated HG alone. Furthermore, the HG/[6]-GR group was almost equal to the DH group. Hence, [6]-GR may enhance the role of HG in the treatment of CHF, for which the HG/[6]-GR couple has better therapeutic efficacy than their single use.

| Histological examination of heart damage
Histological examination of heart damage in Figure 1I Figure 1I).

| Evaluation of the metabolic disturbances in DOX-induced H9c2 cardiomyocyte injury
As metabolomics could comprehensively evaluate the metabolites in cells, the pattern recognition approaches such as "unsupervised" PCA and "supervised" PLS-DA were performed to assess the metabolic disturbances in DOX-induced H9c2 cardiomyocyte injury.  Figure 3B) and PCA data were analysed to evaluate the distributions and the differences between the groups either in negative mode or in positive mode (Figure 2A   in these two modes was also conducted and was shown in Figure   S1. Figure

| Identification of potential biomarkers in CHF treatment and correlation analysis with energy metabolism indicators
Next, potential biomarkers for CHF treatment were identified. indicating the formation of substrates in the mitochondrial energy metabolism ( Figure 3C). In addition, a clustered heatmap and PCA based on the potential biomarkers data were constructed to determine the distributions and find the differences between the groups ( Figure 3B). Taken together, the results demonstrated that the HG/[6]-GR group showed significant therapeutic effects on CHF.
In particular, the therapeutic effect observed for the HG/[6]-GR was much better than that observed in the HG and [6]-GR groups ( Figure 3D-K).

| Related pathways of differential metabolites
MetaboAnalyst 4.0 was used for the detailed pathway analysis, which focuses on exploratory statistical analysis, functional interpretation and advanced statistics for translational metabolomic studies.
Metabolic pathway analysis indicated that eight potential metabolites were important for CHF treatment. As shown in Figure 3A and  also identified as the potential metabolic pathways, which were contributed to energy metabolism ( Figure 3C). The matching status, P value, −log(p) and the impact of each metabolic pathway are listed in Table 2.

| HG/[6]-GR combination promotes mitochondrial respiratory function
To assess whether the observed toxicity of DOX is correlated with mitochondrial respiration defects, a Seahorse XFp analyser was used to determine the OCR and ECAR values of H9c2 cells. DOX specifically impaired both OCR and ECAR values in H9c2 cells ( Figure 4A,B).

| Effect of HG/[6]-GR on the mRNA expression of myocardial microvasculature
As shown in Figure 5A,B, DOX significantly increased the relative mRNA level of ACE and AT1R (P < .01). Conversely, the expression levels of ACE and AT1R in the myocardium of rats in the HG/[6]-GR group were significantly decreased (P < .01). In addition, the results showed that the relative mRNA levels of NRG1 and AngPTL4 in the

| HG/[6]-GR inhibits the expressions of ACE, AGTR1 and NRG1 in myocardial tissue of rats
The expressions of ACE, AGTR1 and NRG1 in cardiomyocytes were determined by immunohistochemical staining. The results showed that ACE, AGTR1 and NRG1 were significantly overexpressed in the DOX group compared with the control group ( Figure 5J). The analysis showed that the expressions of ACE, AGTR1 and NRG1 in the HG and [6]-GR groups were lower than the DOX group. HG/[6]-GR obviously down-regulated the expressions of ACE, AGTR1 and NRG1 versus the DOX group ( Figure 5J).

| D ISCUSS I ON
Pathological mechanism and influencing factors of the occurrence and development of CHF are complex, involving haemodynamics, neurohormones, genetic factors, energy and lipid metabolism, etc.
Professor Neubauer believes that the myocardial energy supply is insufficient or metabolic imbalance during the occurrence of CHF, which leads to the damage of cardiac structure and function thus causes HF. 28 The exhausted heart is referred as "fuelled engines." Professor Van Bilsen 29 proposed the concept of "metabolic remodelling" in view of the occurrence and development of CHF. He believed that the abnormal changes in cardiac structure and function were caused by cardiac metabolic disorders. It is evident that exploring the metabolic pathways of CHF and improving cardiac metabolic disorders become one of the new effective ways to treat CHF. New ways of prevention and treatment of HF are of great benefit.
The neuroendocrine mechanism of CHF is the basis of classical drugs. After obtaining strong evidence to improve the prognosis of patients with HF through carefully designed randomized trials, drugs for the sympathetic nervous system and RAAS have become the pillars of CHF treatment. 30 Therefore, a systematic study of drugs F I G U R E 6 Effects of HG/[6]-GR couple on the relative mRNA and protein expression levels in the heart tissue of rats. (A) Relative LKB1 mRNA level in heart tissue; (B) relative CaMKK2 mRNA level in heart tissue; (C) relative AMPK α1 mRNA level in heart tissue; (D) relative CPT-1 mRNA level in heart tissue; (E) relative Sirt1 mRNA level in heart tissue; (F) relative PGC-1α mRNA level in heart tissue; (G) relative p300 mRNA level in heart tissue; (H) schematic for the role of HG/[6]-GR in LKB1/AMPK α1/Sirt1 signalling pathway in rats; (I) Western blotting images of LKB1, AMPK α1, Sirt1 and PGC-1α; (J) relative LKB1 protein level in heart tissue; (K) relative AMPK α1 protein level in heart tissue; (L) relative Sirt1 protein level in heart tissue; (M) relative PGC-1α protein level in heart tissue. * * P < . AMPK/Sirt1 axis is involved in a variety of biological processes during cell growth. 35 It is noteworthy that the LKB1/AMPK/Sirt1 axis and its downstream targets are highly sensitive targets for DOXinduced cardiac injury. 36 SIRT1 is reported to have a critical role in controlling the cardiac LKB1/AMPK pathway. 37 Activated Sirt1 can also regulate the activity of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and further up-regulate its expression. 38 These signalling pathways are energy-sensing networks and play an important regulatory role in mitochondrial biosynthesis and energy metabolism. 39 In the current study, the results showed that HG/[6]-GR promotes the mRNA and protein expression of LKB1, AMPK α1, SIRT1 and PGC-1α. Then, it can prevent the heart from DOX-induced mitochondrial function disorder.
Although integrated approaches were used to clarify the effectiveness and mechanism of HG combined with [6]-GR in the treat-

| CON CLUS ION
In conclusion, this study demonstrated that HG and This study will provide the theoretical fundament for the further acquaintance of the compatibility theory of Chinese material medical and have a critical significance for the guidance of the clinic practice of TCM.

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

AUTH O R CO NTR I B UTI O N S
Wenjun Zou and Yanling Zhao conceived and designed the study.

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