Hydromorphone hydrochloride preconditioning combined with postconditioning attenuates myocardial ischemia/reperfusion injury in rats by improving mitochondrial function and activating the PI3K/Akt signaling pathway

Thrombolytic therapy or percutaneous coronary intervention for myocardial infarction often cause myocardial ischemia/reperfusion injury (MIRI) and poor prognosis of patients. This study aimed to explore the protective effect and potential mechanism of hydromorphone hydrochloride (HH) on MIRI. Fifty Sprague–Dawley male rats were randomly divided into Sham group, I/R group, HH‐pre group, HH‐post group, and HH‐pre + post group. Except Sham group, MIRI models were established by ligating and relaxing the left anterior descending coronary artery, followed by tail vein injection of HH (0.3 μmol/L) 10 min before ligation (HH‐pre group), 10 min after reperfusion (HH‐post group), and twice at the above two time points (HH‐pre + post group). After intervention, the cardiac function of rats was evaluated by echocardiography, and the levels of myocardial injury markers, oxidative stress indicators, and mitochondrial function indicators were detected. Next, the myocardial infarction area was evaluated by 2,3,5‐triphenyltetrazolium chloride staining, mitochondrial biogenesis, and phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway by western blot. Compared with the I/R group, HH intervention improved cardiac function, decreased myocardial infarction area, reduced serum myocardial injury markers, alleviated oxidative stress, improved mitochondrial function, up‐regulated mitochondrial biogenesis, and activated PI3K/Akt signaling pathway. Moreover, the HH‐pre + post group was superior to the HH‐pre and HH‐post groups in the above aspects. Collectively, HH had protective effect on MIRI rats, and HH preconditioning combined with postconditioning showed optimal efficacy. Such efficacy may be achieved by promoting mitochondrial biogenesis to improve mitochondrial function and reduce oxidative stress, and activating the PI3K/Akt signaling pathway.


| INTRODUCTION
Acute myocardial infarction (AMI) is one of the most common types of ischemic heart disease.Because of its high mortality, AMI has become an important risk factor threatening the life and health of the global population.More than 6 million people worldwide die of AMI every year (Yellon & Hausenloy, 2007).Rapid development of medical technology in recent years allows wide-ranging application of thrombolytic therapy or percutaneous coronary intervention clinically.These two treatments have become the most effective intervention measures to reduce the area of myocardial infarction, improve the clinical symptoms, and thus greatly cut down the risk of death of patients with AMI (Samsky et al., 2021).However, the treatments rapidly recovering the blood flow in myocardial ischemic area may also induce myocardial ischemia/ reperfusion injury (MIRI) (Davidson et al., 2019).MIRI is characterized by severe injury to the ischemic myocardium again following the recovery of blood flow, which can lead to heart failure, ventricular arrhythmia, and increased myocardial infarction area (Hausenloy & Yellon, 2015), and consequently poor prognosis of patients with AMI.Therefore, exploring drugs that can effectively relieve MIRI is of great importance to increase the success rate of heart-related treatments and improve the prognosis of patients with AMI.
Oxidative stress plays a vital role in the pathological process of MIRI.Insufficient blood supply to the myocardial tissue can cause redox imbalance and then oxidative stress.Oxidative stress brings damages to cardiomyocytes, induces inflammatory reactions, and leads to mitochondrial dysfunction (Ayala et al., 2014;Galasso et al., 2021;He et al., 2022).Mitochondrial damage and increased free radicals promote the occurrence of calcium overload (Feissner et al., 2009).Ca 2+ ions in mitochondria react with phosphate-containing compounds to form calcium phosphate.This phenomenon interferes with mitochondrial oxidative phosphorylation, resulting in impaired energy metabolism and reduced ATP production (Bravo-Sagua et al., 2017).Maintaining normal mitochondrial function is essential for preventing oxidative stress (Martin-Fernandez & Gredilla, 2016;Quan et al., 2020).The PI3K/AKT pathway is a critical intracellular signaling pathway to regulate mitochondrial function.This pathway is important in combating MIRI, with main functions of promoting cell proliferation, inhibiting cell apoptosis, suppressing mitochondrial dysfunction, and alleviating inflammatory immune response (Deng & Zhou, 2023;Syed Abd Halim et al., 2023).
Hydromorphone hydrochloride (HH) is a synthetic opioid with more potent sedative and analgesic effects than morphine.Similar to other opioids, the analgesic effect of HH is mainly achieved by binding to μopioid receptors in the center and periphery.HH has been commonly used for postoperative pain, severe trauma pain or cancer pain, and other situations that required effective analgesia (Quigley, 2002).In addition, its protective effect on ischemia/reperfusion injury (IRI) has gradually become an attractive focus in recent years.Kim et al. discovered that HH reduced the level of reactive oxygen species (ROS) in the brain of rats, thereby alleviating brain IRI caused by total intravenous anesthesia (Kim et al., 2016).Xie et al. also revealed that HH effectively inhibited the inflammatory immune response and oxidative stress response caused by IRI in the brain of mice, thereby reducing neuronal apoptosis in hippocampus and improving cognitive ability (Xie et al., 2018).We previously found HH postconditioning could effectively inhibit oxidative stress response and alleviate myocardial injury in rats with MIRI (Qiu et al., 2020).However, the specific mechanism of HH remains to be explained.Hence, a rat model of MIRI was established in this study to explore the protective effect and potential mechanism of HH on MIRI.The results of this study contributed to clarifying the new pharmacological effects of HH and providing new strategies for the prevention and treatment of MIRI.

| Animals
A total of 50 adult male Sprague-Dawley rats aged 7-8 weeks and weighting 200-300 g were selected as research subjects in this study.The rats were kept in an environment with a 12 h:12 h light-dark cycle, a temperature of 22 ± 2°C, and a humidity of 50%-60%.All rats had free access to food and water.All animal experiments were conducted in strict accordance with Guide for the Care and Use of Laboratory Animals to ensure animal welfare.This study was reviewed and approved by the Animal Ethics Committees of Guangdong Provincial Medical Laboratory Animal Center (C202111-03).

| Animal grouping and intervention
After 1 week of adaptive feeding, the rats were randomly and equally divided into five groups: Sham group, I/R group, HH-pre group, HH-post group, and HH-pre + post group.MIRI rat models were constructed according to the method described in a previous study (Katz et al., 2019).Rats in the I/R group, HH-pre group, HH-post group, and HH-pre + post group were subjected to deep anesthesia by inhaling isoflurane (2%-3% volume) and fixed on the animal operating board in a supine position.The ligation was performed with a 5-0 surgical suture at 2-3 mm from the starting point of the left anterior descending coronary artery.Then, a plastic tube with a length of about 5 mm was passed through the ligation to cut off the blood flow of the left anterior descending coronary artery to the maximum extent.Rats in the Sham group received the same operation, but the suture was only threaded below the point at 2-3 mm from the starting point of the left anterior descending coronary artery without ligation and plastic tube compression.After 30 min of ischemia caused by ligation, the 5-0 surgical suture was gently cut off with an ophthalmic scissors, and the plastic tube was removed.After that, the rats were reperfused for 120 min to simulate MIRI.
Tail vein injection of 0.3 μmol/L of HH was performed 10 min before ligation for rats in the HH-pre group (Qiu et al., 2020) and 10 min after reperfusion for rats in the HH-post group.As for rats in the HH-pre + post group, tail vein injection of 0.3 μmol/L of HH was conducted 10 min before ligation and 10 min after reperfusion, respectively.The experimental process was shown in Figure 1.

| Echocardiographic examination
After surgery, the rats were examined by color Doppler echocardiography immediately, and the following indexes were recorded: (1) Left ventricular ejection fraction (LVEF) = [(left ventricular end-diastolic volume − left ventricular end-systolic volume)/left ventricular enddiastolic volume] × 100%; (2) Left ventricular fractional shortening (LVFS) = [(left ventricular end-diastolic diameter − left ventricular end-systolic diameter)/left ventricular end-diastolic diameter] × 100%; (3) the maximum rate of increase of left ventricular pressure (+dP/dt max); (4) the maximum rate of decrease of left ventricular pressure (−dP/dt max).After echocardiographic examination, the rats were killed by excessive anesthesia.Then, the heart and venous blood samples of rats were collected for subsequent experiments.

| Biochemical test
The collected venous blood samples were centrifuged (1258g, 4°C, 3 min), and the supernatant was collected as serum samples for biochemical test.Levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in the serum samples were measured by Indiko automatic biochemical analyzer (Thermo Fisher Scientific, Waltham, MA, USA, USA).Additionally, the level of cardiac troponin I (cTnl) in the serum samples was determined by commercial cTnl ELISA kit (CSB-E08594r, Cusabio, Beijing, China).
F I G U R E 1 Experimental process.

| Assessment of myocardial infarction area
Referring to previous research (Chi et al., 2019), 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution (Biosharp, Hefei, China) was employed to evaluate the myocardial infarction area through myocardial staining.Briefly, the heart was rinsed with ice saline and stored in a refrigerator at −20°C overnight.On the next day, the heart of each rat was sectioned uniformly and serially along the long axis of the left ventricle with a precooled blade to obtain five coronal sections with a thickness of approximately 2 mm.Next, the coronal sections were incubated in 1% TTC solution at 37°C for 30 min and then fixed in 4% paraformaldehyde.After staining, the red area was surviving myocardial tissue and the white area was myocardial infarction tissue.ImageJ software (NIH, Silver Springs, MD, USA) was applied to quantitatively calculate the proportion of myocardial infarction area based on the formula: proportion of myocardial infarction area = [white area/(white area + red area) × 100%].

| Determination of oxidative stress markers in myocardial tissue
Some myocardial tissue was cut into small sections with ophthalmic scissors and placed into precooled physiological saline solution.After centrifugation (2012g, 4°C, 15 min), the supernatant was collected for subsequent experiments.The protein content of the supernatant was detected using bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China) to correct the level of oxidative stress markers in each sample.Next, commercial superoxide dismutase (SOD) detection kit (19,160, Sigma-Aldrich, St. Louis, MO, USA) was used to detect the SOD level of serum samples by colorimetry.More specifically, SOD was measured by testing its ability to stop a chemical reaction involving a dye called nitroblue tetrazolium, which was usually reduced by another chemical called phenazine methosulphate (Ahmed et al., 2022).
Commercial malondialdehyde (MDA) detection kit (ab118970, Abcam, Cambridge, UK) was adopted to test the MDA level in myocardial tissues by colorimetry.Briefly, MDA reacted with thiobarbituric acid (TBA) to form the red complex of MDA-TBA (Ahmed et al., 2022).Then, the absorbance of the complex at 532 nm was detected by a microplate reader to reflect the level of MDA.
The catalase (CAT) level of myocardial tissues was determined by colorimetry using commercial CAT detection kit (ab83464, Abcam, Cambridge, UK).Specifically, CAT reacted with hydrogen peroxide (H 2 O 2 ) in the kit to generate water and oxygen, while unconverted H 2 O 2 reacted with probes in the kit to generate fluorescent products.The absorbance at 570 nm was checked by a microplate reader to reflect the level of remaining H 2 O 2 .Finally, the CAT level of serum samples was calculated based on the inverse ratio of CAT levels to the absorbance.

| Determination of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in myocardial tissue
The level of reactive oxygen species (ROS) in the supernatant of myocardial tissue was detected by ROS detection kit (S0033S, Beyotime, Shanghai, China).The ROS detection kit contained 2′,7′-Dichlorodihydrofluor escein diacetate (DCFH-DA) fluorescent probe.DCFH could react with ROS to generate fluorescence, and the fluorescence intensity was proportional to the level of ROS.The supernatant of myocardial tissue was incubated with DCFH-DA solution at 37°C for 30 min and then washed with phosphate buffered saline (PBS) for 3 times.Next, the fluorescence intensity at 488 nm was measured by a microplate reader and converted into IU/ mL concentration form.
The level of adenosine triphosphate (ATP) in myocardial tissue supernatant was detected by ATP detection kit (S0026, Beyotime, Shanghai, China).Briefly, the standard samples with different concentrations were prepared, then the value of relative light unit was detected by a microplate reader, and the standard curve was drawn.Subsequently, the concentration of ATP in the supernatant was calculated according to the standard curve.

| Assessment of mitochondrial DNA (mtDNA) and nuclear DNA
Based on real-time quantitative PCR (RT-qPCR), the relative expression of mitochondrial DNA (mtDNA) in rat myocardial tissues was evaluated by NovaQUANT® Mouse Mitochondrial to Nuclear DNA Ratio Kit (Millipore, Billerica, MA, USA).DNA was extracted from frozen myocardial tissue through Nuclear-Mitochondria DNA Extraction Kit (Zeye Biology Co., Ltd., Shanghai, China).Then, the primers in NovaQUANT® Mouse Mitochondrial to Nuclear DNA Ratio Kit targeting two mitochondrial genes (trLEV and 12 s RNA) and two nuclear genes (BECN1 and NEB) were utilized to measure the ratio of mtDNA to nuclear DNA in Quant Studio 6 Flex system (Applied Biosystems, Waltham, MA, USA) to reflect the relative expression level of mtDNA.
The results obtained were normalized according to the mean value of Sham group.

| Statistical analysis
SPSS 22.0 software (IBM Corp. Armonk, NY, USA) was used for statistical analysis.The measurement data conforming to the normal distribution were expressed by the mean ± SD; independent sample t-test was adopted for comparison between two groups and one-way analysis of variance for comparison among multiple comparison.The data that did not conform to the normal distribution were tested by Mann-Whitney U. p < .05indicated a significant difference.

| Hydromorphone hydrochloride improves cardiac function of rats with myocardial ischemia/reperfusion injury
As a noninvasive cardiac examination method, echocardiography can conveniently and effectively evaluate the cardiac function of rats (Velasco et al., 2017).The results of echocardiography showed that compared with the Sham group, LVEF, LVFS, +dP/dt max, and −dP/dt max in the I/R group were significantly decreased (p < .05).In comparison with the I/R group, the above four indexes in the HH-pre, HH-post, and HH-pre + post groups increased significantly (p < .05).Moreover, the HH-pre + post group exhibited much higher LVEF, LVFS, +dP/dt max, and − dP/dt max than the HH-pre and HH-post groups (p < .05)(Figure 2a-d).These results suggested that HH could effectively protect the cardiac function of MIRI rats.

| Hydromorphone hydrochloride relieves myocardial injury in myocardial ischemia/reperfusion injury rats
TTC staining is a simple and convenient method to quantitatively evaluate the extent and severity of myocardial infarction in vitro (Kakimoto et al., 2013).According to the results of TTC staining, there was no myocardial infarction in the Sham group, whereas different degrees of myocardial infarction could be observed in the four modeling groups.Furthermore, compared with the I/R group, the proportion of myocardial infarction area in the HH-pre group, HH-post group, and HH-pre + post group decreased significantly (HH-pre + post < HH-post < HH-pre, p < .05)(Figure 3a,b).Collectively, HH could reduce the myocardial infarction area caused by MIRI in rats, and HH preconditioning combined with postconditioning exhibited the best effect.
Elevated serum levels of CK-MB, cTnl, and LDH are important characteristics of myocardial injury and usually closely associated with the severity of myocardial injury (Apple, 1999).The results of biochemical tests showed that the levels of CK-MB, cTnl, and LDH in the serum of rats in the I/R group were significantly higher than those in the Sham group (p < .05).HH intervention notably decreased the levels of CK-MB, cTnl, and LDH in serum of I/R rats (HH-pre + post < HH-post < HH-pre, p < .05)(Figure 3c-e).The above outcomes suggested that HH could effectively alleviate myocardial injury in MIRI rats, and the combination of HH preconditioning and postconditioning had the best effect.

| Hydromorphone hydrochloride inhibits oxidative stress in myocardial tissue of rats with myocardial ischemia/ reperfusion injury
The levels of oxidative markers (SOD, MDA, and CAT) were detected to evaluate the severity of oxidative stress in myocardial tissue of MIRI rats.The results of biochemical test of myocardial tissue showed that compared with the Sham group, the level of MDA in myocardial tissue of rats in the I/R group increased significantly, whereas the activities of SOD and CAT decreased notably (p < .05).HH intervention significantly decreased MDA levels (HH-pre + post < HH-pre < HHpost, p < .05)and increased SOD and CAT activity of I/R rats (HH-pre < HH-post < HH-pre + post, p < .05)(Figure 4a-c).In a nutshell, HH could effectively reduce the oxidative stress in myocardial tissue of MIRI rats, and the effect of HH preconditioning combined with postconditioning was best.

| Hydromorphone hydrochloride improves mitochondrial function in myocardial tissue of rats with myocardial ischemia/reperfusion injury
The effect of HH intervention on mitochondrial function in myocardial tissue of MIRI rats was further explored.The detection results of ROS and ATP revealed that ROS levels were significantly up-regulated and ATP and mtDNA levels were markedly downregulated in myocardial tissue of I/R rats (p < .05).However, HH intervention significantly decreased ROS levels (HH-pre + post < HH-post < HH-pre, p < .01)and increased ATP and mtDNA levels (HHpre + post > HH-post > HH-pre, p < .01) of I/R rats (Figure 5a-c).Besides, western blot results revealed that the p-AMPK, TFAM, and PGC-1α protein levels of I/R rats were notably up-regulated after HH intervention (HH-pre + post > HH-post > HH-pre, p < .01)(Figure 5d,e).Overall, HH could effectively improve the mitochondrial function and promoted mitochondrial biogenesis in myocardial tissue of MIRI rats, increase the energy produced by mitochondria in myocardial cells, and reduce excessive ROS production caused by mitochondrial dysfunction.

| Hydromorphone hydrochloride activates the PI3K/Akt signaling pathway in myocardial tissue of rats with myocardial ischemia/reperfusion injury
The regulatory effect of HH on the PI3K/Akt signaling pathway was further discussed in this study.The western blot results demonstrated that the ratios of p-PI3K/PI3K and p-Akt/Akt in myocardial tissue of rats in the I/R group were much lower than those in the Sham group (p < .05).Compared with the I/R group, the ratios of p-PI3K/PI3K and p-Akt/Akt in the HHpre group, HH-post group, and HH-pre + post group

| DISCUSSION
Our experimental results demonstrated HH had a significant protective effect against MIRI, as evidenced by its ability to improve cardiac function, reduce the area of myocardial infarction, decrease the level of serum myocardial injury markers, inhibit oxidative stress of myocardial tissue, and improve the mitochondrial function in myocardial cells.The protective effect of HH may be achieved by promoting mitochondrial biogenesis and activating the PI3K/Akt signaling pathway.Moreover, the efficacy of HH preconditioning combined with postconditioning was superior to HH preconditioning or postconditioning alone.Similarity, Liu et al. reported that I/R rats were treated with K-H buffer containing 0.3 μmol/L hydromorphone for 10 min at the initiation of reperfusion, and such treatment strategy may protect isolated rat heart against reperfusion injury via activating P13K/Akt/eNOS signaling (Liu et al., 2018).Instead of using isolated hearts, our study directly simulated the process of cardiac ischemia and reperfusion in vivo, which is closer to a real clinical situation.We not only consolidated the beneficial effects of HH on MIRI, but also provided an enlightening basis for the prevention and treatment of MIRI.
The protective effect of HH on MIRI was researched from three aspects in this study.First, the cardiac function of MIRI rat models was evaluated through echocardiography.LVEF, LVFS, and +dP/dt max are essential indexes to evaluate cardiac systolic function, and − dP/dt max can reflect myocardial diastolic function (Lainchbury & Redfield, 1999).HH intervention significantly improved the above four indexes in MIRI rats in our study, indicating that HH could effectively improve the systolic and diastolic function of myocardial tissue.Second, the changes of myocardial infarction area in MIRI rat models were directly observed.Enlarged myocardial infarction area is an important feature of MIRI.Metabolite accumulation and reduction in energy reserves occur in myocardial cells during the ischemic stage, and oxygen and nutrients enter myocardial cells once blood supply is restored.As a result, oxidative stress and inflammatory response are triggered, further inducing myocardial cell necrosis and apoptosis, and ultimately resulting in the enlargement of myocardial infarction area again (Davidson et al., 2019).Therefore, the area of myocardial infarction can reflect the severity of MIRI.We found HH intervention significantly reduced the proportion of myocardial infarction area of myocardial tissue in MIRI rats, indicating that HH could inhibit the enlargement of myocardial infarction area caused by MIRI.Third, the levels of serum markers related to myocardial injury in MIRI rats were detected.HH intervention significantly reduced the levels of CK-MB, cTnl, and LDH in serum of MIRI rats in our study.CK-MB, cTnl, and LDH are abundant in myocardial cells, and in case of MIRI, these three proteins/ enzymes are released from the injured myocardial cells to the peripheral blood to cause an increase in their levels (Apple, 1999).Therefore, the decreased levels of CK-MB, cTnl, and LDH indicate that HH can alleviate the damage of myocardial cells.Based on the comprehensive analysis of the above three aspects, this study powerfully confirmed the protective effect of HH on MIRI.
We further explored its mechanism after confirming the curative effect of HH on MIRI.Oxidative stress is considered a dominant factor that induces cardiac dysfunction and increases myocardial infarction area in the pathological process of MIRI (Zhao et al., 2017).We discovered that HH intervention could increase the level of antioxidant enzymes and decrease the level of lipid peroxide products in myocardial tissue of MIRI rats.Such finding suggested that HH could effectively inhibit the oxidative stress in MIRI.Oxidative stress refers to cell damage caused by excessive accumulation of ROS in cells, and mitochondria are the main sit of ROS production (Zhao et al., 2017).Abnormally increased ROS and mitochondrial dysfunction constitute a vicious circle of interaction in the pathological process of MIRI.During the ischemic stage of MIRI, insufficient oxygen supply leads to the blockage of electron transfer in the mitochondrial electron transfer chain and consequently massive accumulation of electrons.Oxygen concentration increases substantially in mitochondria in the reperfusion phase of MIRI, allowing the restart of the electron transfer chain.Thus, the previously accumulated electrons bind to oxygen molecules and are converted into ROS (Marin et al., 2021).However, excessive ROS will open the transmembrane channel called "permeability transition pore" in the inner membrane of mitochondria and lead to the leakage of substances in the membrane, thereby destroying the normal electron transport chain of mitochondria and inducing mitochondrial apoptosis (Bauer & Murphy, 2020).We found that HH intervention up-regulated the ATP level and downregulated the ROS level in myocardial tissue of MIRI rats.Such outcome indicated that HH could restore the normal electron transport chain of mitochondria, thereby reducing the production of ROS and alleviating oxidative stress.
In addition, we further explored the pathway that HH improved mitochondrial function in myocardial tissue of MIRI rats.Mitochondrial biogenesis, an important factor in maintaining mitochondrial function, refers to the division and subsequent transformation of old mitochondria into new mitochondria.Old mitochondria are unable to produce enough energy and only produce a large amount of ROS.Unlike old mitochondria, new mitochondria can promote oxidative phosphorylation more effectively.New mitochondria can also reduce electron leakage to inhibit ROS production, and effectively convert oxygen and nutrients into ATP for energy supply in cells (Bouchez & Devin, 2019).p-AMPK, TFAM, and PGC-1α are all key factors in the classical pathway of mitochondrial biogenesis, and increased levels of these factors can promote the formation of new mitochondria (Dorn 2nd et al., 2015;Valero, 2014).p-AMPK activates PGC-1α, promoting the interaction between PGC-1α and other proteins and thus up-regulating TFAM expression.Increased TFAM expression contributes to the replication and transcription of mtDNA, and ultimately encourages the production of new mitochondria (Valero, 2014).According to our experimental results, HH intervention increased the level of mtDNA reflecting the number of mitochondria and up-regulated the levels of key regulatory factors p-AMPK, TFAM, and PGC-1α in myocardial tissue of MIRI rats.The results suggested that HH effectively promoted the process of mitochondrial biogenesis.Hence, we believe one of the pathways in which HH relieves MIRI is to improve mitochondrial function and reduce oxidative stress by promoting mitochondrial biogenesis.
The PI3K/Akt signaling pathway is one of the most important signal transduction pathways in the body, which can regulate the mitochondrial function.The activation of multiple downstream targets of PI3K/Akt signaling pathway can reduce the opening of the mitochondrial permeability transition pore and regulate mitochondrial energy production, thereby scavenging intracellular ROS and inhibiting apoptosis, and other subsequent responses.PI3K is the starting point of the PI3K/Akt signaling pathway.When cells receive related signals, PI3K is phosphorylated and activated to catalyze the conversion of phosphatidylinositol diphosphate (PIP2) on the cell membrane into phosphatidylinositol triphosphate (PIP3).The generated PIP3 acts as the second messenger, and 3-phosphoinositide-dependent protein kinase 1 (PDK1) and Akt protein are recruited to the cell membrane.This allows PDK1 to phosphorylate AKT protein, and activated AKT to function by further activating the downstream regulatory pathway (Carnero & Paramio, 2014).Several studies have claimed that activating the PI3K/Akt signaling pathway can inhibit the necrosis and apoptosis of myocardial cells during MIRI through various pathways, thereby effectively alleviating the adverse consequences caused by MIRI (Guan et al., 2020;Xin et al., 2020).In our research, after intervention with HH, the levels of p-PI3K and p-Akt in myocardial tissue of MIRI rats increased significantly, indicating that HH could effectively activate the PI3K/Akt signaling pathway.Therefore, we believed that another pathway for HH alleviating MIRI is to activate the PI3K/ Akt signaling pathway.
Interestingly, we discovered that most of indexes in the HH-post group were slightly better than those in the HHpre group, although the difference was not statistically significant.This may suggest better protective effect of HH postconditioning than HH preconditioning on MIRI.C Penna et al. demonstrated that the first few minutes of reperfusion was the key stage to alleviate MIRI (Penna et al., 2008).Both preconditioning (before ischemia) and postconditioning (at the beginning of reperfusion) using endogenous cardioprotective agents, such as nitric oxide, hydrogen sulfide, endothelin, and bradykinin, can protect myocardial tissue.However, postconditioning is more effective than preconditioning in reducing oxidative damage, inhibiting inflammatory response, and up-regulating the PI3K/Akt signaling pathway (Penna et al., 2015).Such conclusion is basically consistent with the findings of our study.Additionally, most of indexes in the HH-pre + post group were significantly better than those in the HH-pre group and HH-post group.Such outcome suggested that the protective effect of HH was cumulatively and preconditioning combined with postconditioning could exert the best anti-MIRI effect.However, further experiments are needed to confirm whether the combination effect is due to the different treatment or dose doubling.
There are also some limitations in this study.First, in the pathological process of MIRI, the PI3K/Akt signaling pathway regulates various downstream molecules including mTOR, GSK-3β, Bcl-2, and NF-κB and exerts different functions based on different downstream molecules activated (Fulda, 2014).However, this study did not explore which downstream molecules were mainly regulated by the PI3K/Akt signaling pathway activated by HH intervention.Second, neither the number nor the morphological changes of mitochondria in myocardial cells were directly observed in this study, so there was no intuitive evidence to support the view of mitochondrial biogenesis.

| CONCLUSION
To sum up, HH had protective effect on MIRI, and such protective effect may be achieved by promoting mitochondrial biogenesis to improve mitochondrial function and reduce oxidative stress as well as by activating the PI3K/ Akt signaling pathway.Moreover, the combination of HH preconditioning and postconditioning showed better efficacy than preconditioning or postconditioning alone.This study not only clarifies the new pharmacological mechanism and clinical application direction of HH, but also provides new insights for the prevention and treatment of MIRI.

F
Hydromorphone hydrochloride improves cardiac function of rats with myocardial ischemia/reperfusion injury.(a-d) Echocardiography was used to examine LVEF (a), LVFS (b), +dP/dt max (c), and −dP/dt (d) of rats in each group.Statistical analysis was performed using one-way analysis of variance, n = 10, **p < .01 vs. Sham group; # p < .05,## p < .01 vs. I/R group; & p < .05,&& p < .01 vs. HHpre group; ^p < .05,^^p < .01 vs. HH-post group.LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; +dP/dt max, the maximum rate of increase of left ventricular pressure; −dP/dt, the maximum rate of decrease of left ventricular pressure.climbedsignificantly, with the highest ratios in the HH-pre + post group (p < .05)(Figure6a,b).The above findings indicated that HH could activate the PI3K/Akt signaling pathway in myocardial tissue of MIRI rats, and HH preconditioning combined with postconditioning had the best effect.

F
Hydromorphone hydrochloride improves myocardial injury of rats with myocardial ischemia/reperfusion injury.(a) Representative images of myocardial tissue after TTC staining; (b) The proportion of myocardial infarction area in each group of rats; (c-e) Biochemical tests were used to measure levels of CK-MB (c), cTnl (d), and LDH (e) in serum of rats in each group.Statistical analysis was performed using one-way analysis of variance, n = 10, **p < .01 vs. Sham group; ## p < .01 vs. I/R group; && p < .01 vs. HH-pre group; ^^p < .01 vs. HH-post group.CK-MB, creatine kinase-MB; cTnl, cardiac troponin I; LDH, lactate dehydrogenase; TTC, 2,3,5-triphenyltetrazolium chloride.F I G U R E 4 Hydromorphone hydrochloride inhibits oxidative stress in myocardial tissue of rats with myocardial ischemia/reperfusion injury.(a-c) Detection for levels of MDA (a) and activities of SOD (b) and CAT (c) in myocardial tissue of rats in each group.Statistical analysis was performed using one-way analysis of variance, n = 10, **p < .01 vs. Sham group; # p < .05,## p < .01 vs. I/R group; && p < .01 vs. HH-pre group; ^^p < .01 vs. HH-post group.CAT, catalase; MDA, malondialdehyde; SOD, superoxide dismutase.

F
Hydromorphone hydrochloride ameliorates mitochondrial function in myocardial tissue of rats with myocardial ischemia/ reperfusion injury.(a-c) Detection for the levels of ROS (a) and ATP (b) and relative content of mtDNA (c) in myocardial tissues of rats in each group; (d) Representative western blot images of proteins related to mitochondrial biogenesis; (e) Semi-quantitative results of mitochondrial biogenesis-related proteins in rats of each group.Statistical analysis was performed using one-way analysis of variance, n = 10, **p < .01 vs. Sham group; ## p < .01 vs. I/R group; && p < .01 vs. HH-pre group; ^^p < .01 vs. HH-post group.ATP, adenosine triphosphate; mtDNA, mitochondrial DNA; ROS, reactive oxygen species.QIU et al.