A possible new activator of PI3K‐Huayu Qutan Recipe alleviates mitochondrial apoptosis in obesity rats with acute myocardial infarction

Abstract Obesity, which has unknown pathogenesis, can increase the frequency and seriousness of acute myocardial infarction (AMI). This study evaluated effect of Huayu Qutan Recipe (HQR) pretreatment on myocardial apoptosis induced by AMI by regulating mitochondrial function via PI3K/Akt/Bad pathway in rats with obesity. For in vivo experiments, 60 male rats were randomly divided into 6 groups: sham group, AMI group, AMI (obese) group, 4.5, 9.0 and 18.0 g/kg/d HQR groups. The models fed on HQR with different concentrations for 2 weeks before AMI. For in vitro experiments, the cardiomyocytes line (H9c2) was used. Cells were pretreated with palmitic acid (PA) for 24 h, then to build hypoxia model followed by HQR‐containing serum for 24 h. Related indicators were also detected. In vivo, HQR can lessen pathohistological damage and apoptosis after AMI. In addition, HQR improves blood fat levels, cardiac function, inflammatory factor, the balance of oxidation and antioxidation, as well as lessen infarction in rats with obesity after AMI. Meanwhile, HQR can diminish myocardial cell death by improving mitochondrial function via PI3K/Akt/Bad pathway activation. In vitro, HQR inhibited H9c2 cells apoptosis, improved mitochondrial function and activated the PI3K/Akt/Bad pathway, but effects can be peripeteiad by LY294002. Myocardial mitochondrial dysfunction occurs following AMI and can lead to myocardial apoptosis, which can be aggravated by obesity. HQR can relieve myocardial apoptosis by improving mitochondrial function via the PI3K/Akt/Bad pathway in rats with obesity.


| INTRODUC TI ON
As a severe disease, acute myocardial infarction (AMI) is responsible for cardiac insufficiency and even cardiac structure impairment. 1 Moreover, AMI is not only one of the most dangerous coronary heart diseases but is also an important factor for cardiac insufficiency; it can induce death in critical patients. 2 A decline in contractility and a large increase in myocardial infarction are the major pathophysiological manifestations observed after AMI. 3 In this context, this study proposes a new approach for preventing AMI and facilitating the treatment of AMI.
Increases in obesity, hyperuricemia, diabetes and hyperlipidaemia (HLP) are often accompanied by obesity, which can induce hypertension and insulin resistance. Both can be seen as chronic hyper-inflammatory states. 4 A significant predisposing factor HLP often occurs with AMI. 5 After AMI, the infarct size can be increased by acute mixed HLP. 6 HLP is a highly specific risk factor for cardiovascular disease. 7,8 The pathobiological value of HLP in AMI patients has yet to be determined.
Mitochondrial dysfunction plays an important role in AMI. 9,10 In Asian countries, the treatment of heart disease, including angina, chronic heart failure and myocardial infarction, widely employs traditional Chinese medicine (TCM). As the Chinese herbal compound, Huayu Qutan Recipe (HQR) primarily consists 9 herbal compounds. Our previous study has shown that HQR regulates lipid metabolism via the SREBP-2 signal pathway. 11 However, studies on HQR myocardial protection in rats with obesity have rarely been reported. The current study focuses on evaluating AMI-induced mitochondrial dysfunction to better reduce myocardial injury by using HQR, as well as to cross the gist between mitochondrial dysfunction and myocardial cell apoptosis in order to identify new therapeutic targets and examine the correlated underlying mechanisms.

| Chemicals and reagents
Huayu Qutan Recipe granules were provided by Sichuan New Green Pharmaceutical Co., Ltd in China. Huayu Qutan Recipe granules were dissolved in heated deionized water (60°C) to obtain stock solutions with different concentrations (0.45, 0.9 and 1.8 g/ml). The stock solutions were preserved in refrigerator (4°C).
A total of 60 male rats were divided into 6 groups, as follows: sham-operated group, AMI group, AMI (obese) group, the groups fed with 4.5 g/kg/d HQR, 9.0 g/kg/d and 18.0 g/kg/d HQR. Each group included 10 rats. All rats in each group were fed with general maintenance feed for 2 weeks to conform to the controlled environment. The rats in the AMI and sham groups were raised on general maintenance feed for 8 weeks, whereas those in the other 4 groups were raised on a high-fat diet (HFD). Three groups-the sham group, the AMI group and the AMI (obese) group-received intragastric infusions with deionized water. The other 3 groups were received intragastric infusions of HQR with different concentrations (4.5, 9.0 and 18.0 g/kg/d) for 2 weeks before AMI. The components of HFD were as follows: 13% fibre, 44% carbohydrate, 11% unsaturated fat, Program Project-Industrial Major Project-Innovative research and development of a large variety of traditional Chinese medicine Yuxuebi preparations for the treatment of rheumatoid arthritis and heart disease (2020JH1/10100022). activated the PI3K/Akt/Bad pathway, but effects can be peripeteiad by LY294002.
Myocardial mitochondrial dysfunction occurs following AMI and can lead to myocardial apoptosis, which can be aggravated by obesity. HQR can relieve myocardial apoptosis by improving mitochondrial function via the PI3K/Akt/Bad pathway in rats with obesity.

K E Y W O R D S
acute myocardial infarction, apoptosis, Huayu Qutan Recipe, mitochondrial dysfunction, obesity, PI3K/Akt/Bad pathway 25% total fat containing 18% protein, ash and other ingredients. 12 The rats with a 30% increase in body weight indicated that the obesity model was successfully established and thus can be selected for further study. 13

| Surgical procedure
Rats were anaesthetized (isoflurane via inhalation anaesthesia), then to assess for anaesthetic effects, the rats were paw-pinched and tail-pinched. The AMI model was established as described in a previous report. 14 A left thoracotomy through the fifth intercostal space was performed to completely reveal the heart. The left anterior descending branch of the heart was permanently ligated with a 6-0 silk suture.

| Detecting blood fat, myocardial enzyme spectrum and inflammatory factor in serum
Blood was extracted from the abdominal aorta to detect the lev-

| Detecting cardiac function
Noninvasive transthoracic echocardiography (VisualSonics of Vevo2100) was employed to assess the left ventricle morphology and function of anaesthetized rats (Matrx VIP 3000). The mean included a two-dimensional mode, including blood flow detection with the pulsedmode Doppler and the time-motion ultrasound. The left ventricular end-diastolic internal diameter (LVIDd) and left ventricular end-systolic internal diameter (LVID), left ventricular fractional shortening (FS) and left ventricular ejection fraction (EF) were measured and calculated. 15

| Myocardial infarction area measurement
After successful AMI angioplasty, 5 ml of blood was drawn from the caudal vein, and 2 ml of Evans blue dye (0.5%) was injected into the carotid artery. After the dye was distributed to the whole-heart tissue (out of the blood supply area of the anterior descending branch in the left coronary artery), the rat was anaesthetized, and the heart was quickly removed. The ischaemic area, infarct area and normal area were observed. After the heart was fixed with 4% paraformaldehyde and restained with 1% red tetrazolium for 15 min, the ischaemic part appeared red, the infarcted part turned white, and the normal part was blue. The degree of infarction was determined using the following formula: the degree of infarction = (infarct/ischaemic area) × 100%.

| Histological evaluation of myocardial tissue via haematoxylin-eosin staining
Cutting myocardial tissues (non-necrotic part) were sliced into thin sections with a thickness of 5 μm and then haematoxylin-eosin (HE)stained after paraffin embedding as described in a previous study. 16 The myocardial tissues were infiltrated for 24 h with 4% paraformaldehyde and then diverted to 70% ethanol. The myocardial tissues were observed by light microscopy. 17 The degree of myocardial damage was evaluated on a scale of 0 to 4, as follows: 0 (normal), preserved normal myocardial tissues; 1 (minor damage), localized necrosis and interstitial oedema; 2 (moderate damage), extensive myocardial cell necrosis and swelling; 3 (severe damage), neutrophil infiltration, compressed capillaries, and necrosis with contraction bands; and 4 (highly severe damage), compressed capillaries and haemorrhaging, neutrophil infiltration, and diffuse necrosis with contraction bands. 18

| SOD and MDA in myocardial tissue
The myocardial tissue was placed on an ice table, and the normal myocardial tissue (5-10 mg) around the necrotic area was removed. The myocardial tissue was weighed and then crushed with scissors and a homogenizer. The 5%-10% tissue homogenate (5-10 ml) was prepared by adding normal saline. In accordance with the instructions provided in the reagent kit, the tissue homogenate and reagent were mixed in a centrifuge tube and then cooled immediately at 95°C after being taken out (40 min). The supernatant was centrifuged for 10 min at 3500-4000 × g. The supernatant was taken out at 532 nm with 1 cm light diameter. The distilled water was zeroed, the absorbance was measured, and the malondialdehyde (MDA) was measured based on the absorbance value. The prepared tissue homogenate was mixed with corresponding reagents as specified in the instructions provided with the reagent kit, and the superoxide dismutase (SOD) was determined at 550 nm.

| Observation of mitochondria by electron microscopy
After being thinly sliced (1 mm 3 small volume), the myocardial tissues were collected immediately. The tissues were fixed in 2% glutaraldehyde in an environment at 4°C. They were washed with pH 7.4 phosphate-buffered saline (0.1 mol/L) and then fixed in 1% osmium tetroxide by using 1% aqueous uranyl acetate to stain. The medium was embedded with capsules, and the specimens were placed on the slide for about 48 h (70°C). Alkaline lead citrate and uranyl acetate were used to stain the sections, which were then visualized by electron microscopy (Hitachi/H-7650, Japan).

| Using TUNEL to assess apoptosis in the myocardial tissues
Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabelling (TUNEL) was conducted to evaluate apoptosis by using the In Situ Cell Death Detection Kit (Solarbio). Paraffin sections were used for TUNEL staining. 16 After deparaffinization, the paraffin sections were incubated with 10 μg/ml of proteinase K and then rehydrated for 15 min. Fresh TUNEL reaction mixtures were added to sections, which were incubated for 60 min in darkness and at 37°C.
Myocardial tissues (10 mg) were added to the reaction buffer and then incubated for 2 h at 37°C. The enzyme-catalysed release was quantified using a fluorimeter (405 nm).

| Preparation of the mitochondrial suspension
The rats were executed as described in a previous study. 19 The heart was removed and placed in a breaker with a pH 7.4 ice-cold isolated buffer (1 mM EDTA, 250 mM sucrose and 10 mM Tris-HCl). After the tissue samples were trimmed, they were rinsed using a homogenizer in an isolation buffer and then taken out (50-100 mg). The entire process was performed at 4°C to maintain mitochondrial integrity. Centrifugation (700 × g) was conducted for 10 min, and the supernatant was again collected for centrifugation (7000 × g) for 10 min. The supernatant was then discarded. Resuspension was conducted to wash the mitochondrial pellets (5 ml isolated buffer) and centrifuge (7000 × g) then twice for 10 min. The clean mito-

| RNA extraction and cDNA synthesis and real-time qPCR
The Trizol Reagent (Invitrogen) was used to isolate the total genome RNA, the quality of which was evaluated at 260 nm by spectrophotometry. Reverse transcription was conducted using 1 µg of total RNA and M-MLV Reverse Transcriptase Kit (Promega A3500). A 40 µl total reaction system was used in a Thermal Cycler with 96 wells (Applied Biosystems) in accordance with the following reactive processes: 72°C for 3 min, 42°C for 90 min and 70°C for 15 min. Preservation at 4°C followed. The copy number of the target gene transcription level with cDNA templates was examined by RT-qPCR. PCR (QIAGEN) using SYBR Premix Ex TaqII (TakaraBioINC) was operated with the Rotor-Gene Q detection system 24 in a 20 µl setup (SYBR Premix Ex Taq II 10 µl + synthetic cDNA 1 µl + primers 0.5 µM), with the following steps: 95°C for 10 min; 95°C for 10 s, 40 cycles, 60°C for 15 s; 72°C for 20 s; and 72°C for 10 min. The value was determined, and GAPDH was chosen as the inner control. 25 The sequences of the PCR primers (two pairs) employed in this study are listed in Table 1.

| Preparation and compound analysis of HQRcontaining serum
Male SD rats were intragastrically treated with normal saline (control) or HQR (9.0 g/kg/d) once daily for 7 days. Two hours after the last administration, blood was collected from the aorta ventralis, stored at 4°C for 1 h and centrifuged at 2000 rpm/min for 30 min. Serum samples from the same group were pooled, inactivated in a 56°C water bath for 30 min and sterilized by filtration. The HQR-containing serum was stored at −80°C for subsequent vitro experiments.

Target Gene
Primer Sequence Size (bp)

| Assignment of cell groups
Cells were divided into four groups as follows: control group,

| Electron microscopy
Cells were fixed in 4% glutaraldehyde at 4°C for approximately 2 h, washed with 0.1 M sodium dimethyl arsenate three times and centrifuged between the washing steps. The remainder of the protocol was similar to that of the in vivo experiment.

| Preparation of mitochondrial suspension and assessment of mitochondrial function in vitro
Mitochondria were isolated using the Mitochondrial Isolation Kit (Beyotime) and maintained on ice until use. Mitochondria were resuspended in buffer to achieve a protein concentration of 5 mg/ ml. Cells (1 × 10 5 cells/ml) were plated in 6-well plates, incubated at 37°C for 20 min and centrifuged at 600 × g for 3-4 min. The supernatant was discarded. Thereafter, cells were centrifuged as described. The supernatant was discarded. Lastly, cells were stained with 1× JC-1 staining buffer and centrifuged as described.
The supernatant was discarded. MMP and mPTP were visualized according the manufacturer's instructions. 19,29 All steps were carried out at 4°C.

| Determination of protein concentrations
Cells were harvested, and proteins were extracted from cells in a lysis buffer. The protein concentration was measured using the BCA protein assay.

| Statistical analysis
Statistical analysis was performed using SPSS 17.0 Software (SPSS Inc.). Data were expressed as the mean ± standard error. One-way analysis of variance (ANOVA) was used to compare 4/5 independent groups. The two-to-two comparison among groups was used to analyse the variance The LSD-t test was used to compare multiple comparisons between different groups. We regarded p < 0.05 represent having statistically difference.

| Chemical components of HQR
The

| HQR improved blood fat, myocardial enzymes, cardiac function and inflammatory factors after AMI
Blood fat composition is shown in Figure 2A, cardiac function in Figure 2B, myocardial enzymes in Figure 2C, inflammatory factors in Figure 2D and infarction size in Figure 2E to further evaluate the standard of myocardial injury and the protective effect and mechanism of HQR.
The TC, LDL-C and TG increased in the AMI (obese) group relative to that in the AMI and sham groups (p < 0.05). Their values decreased via pretreatment with HQR. HDL-C decreased in the AMI (obese) group relative to the HDL-C levels in the AMI and sham groups (p < 0.05), which increased via pretreatment with HQR ( Figure 2A). The EF and FS decreased in the AMI and AMI (obese) groups (particularly with a large increase in the AMI (obese) group), relative to those in the sham group (p < 0.05). The LVIDs and LVIDd values increased in the AMI and AMI (obese) groups (markedly increased in the AMI (obese) group) relative to those in the sham group (p < 0.05). The values increased via pretreatment with HQR ( Figure 2B). The cTNI and CK-MB values increased in the AMI and AMI (obese) groups (particularly with a large increase in the AMI (obese) group), relative to those in the sham group (p < 0.05), which were decreased via pretreatment with HQR ( Figure 2C). The IL-1β and TNFα values increased in the AMI and AMI (obese) groups (markedly increased in the AMI (obese) group), relative to those in the sham group (p < 0.05), which were decreased via pretreatment with HQR ( Figure 2D). The infarction size (%) increased in the AMI and AMI (obese) groups, relative to that in the sham group (p < 0.05), which was not decreased via pretreatment with HQR ( Figure 2E).

| HQR improved the imbalance between oxidation (MDA) and antioxidation (SOD) after AMI
The MDA levels in the AMI and AMI (obese) groups were higher than those in the sham group (p < 0.05). However, the high MDA level could be decreased via pretreatment with HQR. The SOD levels in the AMI and AMI (obese) groups were lower than the SOD level in the sham group (p < 0.05). However, pretreatment with HQR could increase SOD. (Figure 2F).

| HQR improved the pathological structure of the myocardium after AMI
We evaluated the myocardial protective effect of HQR after AMI by HE staining of myocardial tissues ( Figure 2G). Considerably disorganized myocardial cells (interstitial oedema) were observed in the AMI and AMI (obese) groups, which was remedied via pretreatment with HQR, as indicated by the alleviation of both interstitial oedema and disorganized myocardial cells. The total damage was evaluated using a histological score ( Figure 2G).
Statistical results indicated that the myocardial tissue damage in the AMI and AMI (obese) groups increased relative to that in the sham group (p < 0.05); the myocardial tissue damage was higher in the AMI (obese) group than in the AMI group (p < 0.05). All myocardial tissue damage scores decreased in the HQR groups (p < 0.05) ( Figure 2G).

| HQR relieves cell apoptosis after AMI
The TUNEL assay was used to determine the protective effect of HQR on myocardial tissue cell apoptosis after AMI in rats with obesity ( Figure 4A). The number of apoptotic cells in myocardial tissues in the AMI and AMI (obese) groups increased relative to that in the sham group (p < 0.05). However, pretreatment with HQR could reduce the number of myocardial tissues apoptotic cells ( Figure 3A).
The caspase-9/3 activity in myocardial cells in the AMI and AMI (obese) groups was higher than that in the sham group (p < 0.05).
However, pretreatment with HQR decreased the caspase-9/3 activity of the myocardial cells ( Figure 3B,C). Western blot analysis was performed to assess the protein levels of cleaved caspase-9/3 ( Figure 3D). The protein levels of cleaved caspase-9/3 in the AMI and AMI (obese) groups increased relative to that in the sham group (p < 0.05). However, pretreatment with HQR reduced the mRNA levels of caspase-9/3 and protein levels of cleaved caspase-9/3 (p < 0.05) ( Figure 3D).

| Morphological changes in myocardial mitochondria after AMI
Using an electron microscope, we evaluated the morphological changes in myocardial mitochondria to determine the myocardial damage and the protective effect of HQR. (Figure 3B).
Electron microscopy images (10,000× and 40,000×) revealed abnormal morphological mitochondria in the cells of the myocardial tissues in the AMI and AMI (obese) groups. The membrane appeared swollen and ruptured (denoted by paired yellow arrows) after AMI.

F I G U R E 4 Huayu Qutan recipe (HQR) decreased Bax and increased Bcl-2 (Immunofluorescence results). The expression of Bax and
Bcl-2 in mRNA (A) and protein (B) level. Data are shown as mean ± SD. *p < 0.05 versus sham group, # p < 0.05 versus AMI (obese) group, Δ p < 0.05 versus HQR (4.5 g/kg.d) group, ▲ p < 0.05 versus HQR (9.0 g/kg.d) group. (n = 3). Immunofluorescence results of Bax (green) and Bcl-2 (red) expression in nephridial tissue (C), the scale bars represent a length of 500 μm on histology Meanwhile, the sham group revealed normal morphological mitochondria (denoted by a single yellow arrow). The number of abnormal morphological mitochondria increased in the AMI and AMI (obese) groups (markedly increased in the AMI [obese] group) than in the sham group (p < 0.05). Pretreatment with HQR decreased the number of abnormal morphological mitochondria (p < 0.05) ( Figure 5C).

| HQR modulates the PI3K/Akt/Bad pathway in vivo
We The mRNA expression of PI3K, Bad and Akt decreased in the AMI group (markedly reduced in the AMI [obese] group), relative to that in the sham group (p < 0.05). The mRNA expression of these genes could be increased via pretreatment with HQR (p < 0.05) ( Figure 7A).
Western blot analysis of target genes in the PI3K/Akt/Bad pathway showed consistent results in protein levels ( Figure 7B). HQR promoted Bad and Akt phosphorylation (i.e. increased the p-Bad/Bad and p-Akt/Akt ratios) ( Figure 7B).

| HQR improved PA + hypoxia-induced mitochondrial dysfunction in vitro
To further assess the protective effects of HQR-containing serum,    Figure 9A).
Compared to the control group, the ROS levels increased in the hypoxia and PA + hypoxia groups (p < 0.05), cells pretreated with 2, 4 and 8% HQR-containing had an decreased ROS levels (p < 0.05) ( Figure 9D). As such, 8% HQR-containing serum was selected for subsequent experiments.

| HQR modulates the PI3K/Akt/Bad pathway to inhibit apoptosis and mitochondrial dysfunction in vitro
To investigate whether the anti-apoptotic effects of HQR-containing serum were associated with the PI3K/Akt/Bad pathway, LY294002  Figure 10A). Compared to the control group, the MMP level (p < 0.05) was decreased but the mPTP level (p < 0.05) was increased in the hypoxia and PA + hypoxia groups. Compared with the hypoxia and PA + hypoxia groups, the MMP level (p < 0.05) was increased but the mPTP level (p < 0.05) was decreased after HQR and LY294002 treatment can reverse the effects of HQR ( Figure 10B,C). Compared to the control group, the ROS levels increased in the hypoxia and PA + hypoxia groups (p < 0.05), cells pretreated with HQR can decrease ROS levels and LY294002 treatment can reverse the effects of HQR (p < 0.05) ( Figure 10D).

| DISCUSS ION
As the leading cause of death worldwide, ischaemic heart disease (IHD) leads to more than 9 million deaths annually. AMI is a manifestation of IHD in which coronary atheromatous plaque ruptures, inducing an acute thrombotic occlusion of the coronary artery and seriously confining or completely obstructing blood flow to myocardial tissues, depriving myocardial cells (namely AMI) of nutrients and oxygen, leading to their death. 9 Hyperlipidaemia can also aggravate myocardial damage. 27 Among the postulated mechanisms for the occurrence and development of AMI, mitochondria have drawn considerable attention. [28][29][30] As the energy powerhouse of the cell, mitochondria produce the largest amount of ATP. 31 After an injury, the mitochondrial membrane potential is reduced. Harmful by-products of ATP, such as proapoptotic factors (Cyt-c) and ROS, are discharged. Cyt-c can exert harmful effects on the function and fate of the cell. 32 Mitochondrial dysfunction can be viewed as a predictive factor for cell death. 33 In the current study, we chose the non-infarcted myocardial tissue because improving and reducing the severity of the injury of noninfarcted myocardial tissue is crucial during clinical treatment.
The major findings in the current study are that myocardial injury is more severe after AMI combined with obesity and that HQR exerts potentially protective effects on myocardial injury induced by AMI. This study also determines and mechanisms of HQR. These  Our study demonstrated that the rats fed with HFD for 8 weeks can overt obesity can be induced rats in the form of weight gain with an accumulation of perirenal fat. HFD can induce inflammation, hyperlipidaemia and oxidative stress (imbalance between oxidation and antioxidation). It can damage the mitochondrial dynamics/biogenesis and the respiratory chain complex enzyme in myocardial tissues in rats. 26 In the present study, hyperlipidaemia can aggravate the damage to the myocardial histological structure in AMI, in contrast to non-hyperlipidaemia. We found that HFD could induce obesity and hyperlipidaemia (increasing LDL-C, TG and TC as well as reducing HDL-C). Using HQR as a hypolipidaemic drug can improve hyperlipidaemia (Figure 2A). We used ELISA to examine IL-1β and TNFα. The results indicated that AMI could induce systemic inflammatory response (elevated concentrations of TNFα and IL-1β), which decreased when pretreated with HQR ( Figure 2D). Different  Figure 2G). Cardiac function can be reduced via the structural injury of the heart ( Figure 2B). Meanwhile, the TUNEL assay revealed that AMI can promote myocardial apoptosis ( Figure 3A). Nonetheless, HQR can decrease the number of apoptotic cells ( Figure 3A). As the principal executors of apoptosis correlated with the final phase, caspase-9/3 activity is a significant indication of apoptosis. Thus, we measured caspase-9/3 activity in the present study. Caspase-9/3 was highly activated in the AMI group, relative to that in the sham (particularly in the group with obesity) (p < 0.05). Meanwhile, the immunofluorescence results for the expression of the apoptosis-related gene Bax and Bcl-2 in the myocardial tissues revealed that AMI increased the Bax expression and decreased the Bcl-2 expression, particularly in the obese group.
AMI has previously been shown to induce mitochondrial dysfunction in myocardial tissue. 9 Mitochondrial apoptotic signalling pathways (the ERK-CREB and ERK1/2 pathways) play critical roles in AMI injury. 46,47 In the present study, AMI could induce an abnormal mitochondrial morphology (swelling and even membrane rupture) and further damaged the mitochondria; moreover, HQR could protect the mitochondria after AMI combined with hyperlipidaemia ( Figure 5). Mitochondrial and redox homeostasis performs a key function in pathophysiology after AMI. An impaired mitochondrial function of myocardial cells and increasing oxidative stress are significant factors influencing AMI damage. 42 Under conditions of ischaemia, lack of oxygen and substrates can repress mitochondrial respiration, and cells must translate to glycolysis, markedly decreasing the capability of cells to produce ATP rapidly. ATP depletion increases the osmotic gradient, which drives water into the mitochondrial matrix to promote cell swelling. 48 In addition, hyperlipidaemia results in the mass production of mitochondrial ROS (hazardous substance), inducing mitochondrial oxidative injury, which activates caspase-dependent podocyte apoptosis via the mitochondrial apoptotic signalling pathway, such as damaged MMP and mitochondrial respiratory function and induced apoptosis. Mitochondrial dysfunction results in myocardial injury. 49,50 In the current study, we built a model of AMI in rats with obesity pretreated with HQR. We determined the MMP, mPAP opening (%), ROS, mtDNA, oxygen consumption rate, RCR, the activity of mitochondrial respiratory chain complex enzymes (Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ) and ATP, which were considered as indicators of mitochondrial function. RCR, MMP, oxygen consumption rate, mitochondrial respiratory chain complex enzymes (Ⅰ, Ⅱ, Ⅲ, Ⅳ and Ⅴ) and ATP were reduced in the AMI and AMI (obese) groups (particularly in the AMI [obese] group); however, the ROS and mPAP opening (%) were increased in the AMI group (markedly increased in the AMI [obese] group). Pretreatment with HQR could improve mitochondrial function ( Figure 5).
The copy number of the mtDNA in each mitochondrion is constant; thus, the mtDNA total copy number can estimate the quantity of mitochondria. 21,51 Although the mechanism of mtDNA injury has yet to be determined, mtDNA is more fragile when exposed to oxidative reaction than the nucleus DNA, considering that mtDNA is near the respiratory chain. In the present study, we examined the damage of AMI on mtDNA (real-time qPCR) by calculating the long/ short fragment ratio (long/short ratio). The long/short fragment ratio was reduced by AMI, but pretreatment with HQR can increase the ratio ( Figure 5).
As an important adaptation of exposure to chronic energy  Normally, mitochondria undergo a dynamic process, including fusion and fission. The dynamic course is important to preserve the shape, size and network of mitochondria. They are controlled by correlated proteins, including Drp1 and Mfns. 53 The results of the present study ( Figure 6) showed that the expression of Mfn1/2 and Drp1 was reversed after AMI, showing a chaotic mitochondrial fission-fusion balance. Mfn1/2 and Drp1 have important functions in mitochondrial fusion and fission, respectively. In this study, Mfn1/2 increased, whereas Drp1 decreased after AMI in non-infarct tissue.
Activation of the PI3K/Akt/Bad signalling pathway function represses apoptosis mediated by mitochondria. 54 The close relationship between them prompted us to evaluate the effect of PI3K. PI3K is a phosphatidylinositol kinase with activities similar to those of a serine/threonine-specific protein kinase and a phosphatidylinositol kinase. 47 After activation, the phosphatidylinositol family members on the cell membrane can be phosphorylated, and the downstream signal molecule Akt can be recruited and activated. The activated Akt then phosphorylates the Ser136/Ser112 residues of the Bad protein. 55 Phosphorylated Bad is separated from the apoptosispromoting complex and forms a 14-3-3 protein complex, thereby inactivating its apoptosis-promoting function, consequently inhibiting apoptosis. 56 The present study showed that HQR effectively regulated the expression of apoptotic PI3K/Akt/Bad pathway-related proteins; moreover, HQR could enhance PI3K and p-Akt expression and downregulate the expression of Cyt-c, cleaved caspase-3 and PAR ( Figure 7). These results showed that HQR could adjust the mitochondrial function and inhibit apoptosis induced by AMI via the activation of the PI3K/Akt/Bad pathway.
In the current study, isolated mitochondria were used, AMI promoted high ROS production, which damaged mtDNA. Ultimately, the mitochondrial respiratory function, biogenesis and dynamic func-

ACK N OWLED G EM ENTS
The authors would like to thank all of the rats, the team of investigators, research partners and operations staff involved in this study.
We want to thank professor Jin-song Kuang and Xian-sheng Meng for scientific and my wife Xiao-lin Jiang for help.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.