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Introduction

Myocardial ischemia-reperfusion (IR) injury is the predominant injury resulting from revascularization treatments, such as coronary thrombolysis, percutaneous coronary interventions, coronary artery bypass grafts and cardiac transplants (1). During ischemia, mitochondria produce reactive oxygen species (ROS) and during reperfusion an extra burst of ROS generation occurs (2). Furthermore, during reperfusion, multiple molecular inflammatory cascades are activated; including those involving interleukin (IL)-1 and tumor necrosis factor (TNF) α. Chemokine production is induced within hours of reperfusion (3), with pro-inflammatory cytokines sequentially inducing cell autophagy (4).

Autophagy is a type of programmed cell death, which has been indicated to be significant in cell homeostasis, as well as cell defense and adaptation to adverse environments (5–7). Autophagy has an important role in the heart and activation of autophagy has been observed in a variety of heart diseases, including cardiac hypertrophy, heart failure and IR injury. Autophagy performs two opposing functions in the heart (8); it has a protective role during nutrient deprivation and other forms of cellular stress, however, excessive autophagy may be used for self-destruction (9).

Ulinastatin is a multivalent enzyme inhibitor, which is predominantly used for the treatment of pancreatitis, severe infection-induced acute circulatory failure, as well as for the prevention of multiple organ failure (10). Recently, ulinastatin has been found to exhibit myocardial protective effects through inhibiting the expression of TNF, reducing levels of oxygen free radicals and increasing levels of endogenous nitric oxide (11,12). During myocardial IR injury, ulinastatin has been reported to show protective effects via the induction of an anti-inflammatory response (1). However, whether the protective effect of ulinastatin in cardiomyocytes is associated with cardiomyocyte autophagy, is yet to be elucidated. The present study aimed to investigate whether ulinastatin reduces cardiomyocyte injury through regulating autophagy and its associated mechanisms.

Material and methods

Animals

All animal experiments were approved by the Animal Research Ethics Committee of the Second Military Medical University (Shanghai, China). The experiments conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

Cell culture and experimental protocols

Neonatal cardiomyocytes were prepared from the hearts of Sprague-Dawley rats (age, <3 days) (13). On day four, the cardiomyocytes were randomly divided into the following three groups: Con, a control group where the cells were cultured in Dulbecco’s modified Eagle’s medium and incubated in an atmosphere of 5% CO2 for 24 h; HR, a hypoxia-reoxygenation group where the cells were incubated with 1% O2, 5% CO2 and 94% N2 for 24 h, followed by 5% CO2 and 95% air for 6 h; and an ulinastatin group, where the cells were treated with 1×104 U/l ulinastatin for 30 min prior to HR, then were incubated under the same conditions as the HR group. In order to investigate whether mammalian target of rapamycin (mTOR) was involved in the protective effect of ulinastatin, the cells were treated with the mTOR inhibitor, rapamycin (20 nM) 30 min prior to ulinastatin treatment.

MTT assay

An MTT assay was used to assess cardiomyocyte vitality. The cardiomyocytes were cultured in 96-well plates at a density of 1×103 cells. MTT solution (10 μl; Sigma, St. Louis, MO, USA) was added to the growing cells and incubated for 4 h. The crystals were then solubilized by adding 100 μl solubilization solution. The absorbance of the purple solution was determined at a wavelength of 450 nm using a microtiter plate reader (Bio-Rad, Hercules, CA, USA).

Western blot analysis

The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according the manufacturer’s instructions. Equal quantities of protein (50 μg) from the cardiomyocytes were subjected to western blot analysis using anti-LC3-I, -LC3-II (Sigma-Aldrich, St. Louis, MO, USA), anti-p-Akt, -p-mTOR and -p-P70S6K (Cell Signaling Technology, Inc., Beverly, MA, USA) primary antibodies to detect LC3, p-Akt (Ser-473), p-mTOR (Ser-2448) and p-P70S6K (Thr-389) protein expression, respectively. An enhanced chemiluminescence detection kit (Amersham Biosciences, Picastaway, NJ, USA) was used to detect the immunoreactive protein bands. The autophagy results were presented as the LC3-II/LC3-I expression ratio.

In vivo rat model and experimental protocols

Sprague-Dawley rats weighing between 250 and 300 g were anesthetized using intraperitoneal injection of 10% chloral hydrate (300 mg/kg body weight), prior to endotracheal intubation. IR was induced by ligating the left anterior descending artery as previously reported (14). Thirty rats were randomly divided into three equal groups: Control group, where the rats underwent thoracotomy without ligation; IR group, where the rats were treated with ischemia for 30 min and reperfusion for 6 h; and an ulinastatin group, where the rats were treated with ulinastatin (1×104 U/kg body weight) by intraperitoneal injection, 30 min prior to IR, followed by the same conditions as the IR group.

Measuring infarct size

Myocardial infarct size was measured as previously described (15). The total left ventricular area, infarct area (INF) and area at risk (AAR) were assessed. The percentage of the INF/AAR was calculated.

Lactate dehydrogenase (LDH) assay

Blood and culture serum were collected following reperfusion from the rats and cultured cardiomyocytes, respectively, in order to determine LDH levels.

Statistical analysis

Quantitative data are presented as the mean ± standard error. Statistical significance was determined using one-way analysis of variance and P<0.05 was considered to indicate a statistically significant difference.

Results

Ulinastatin attenuates HR-induced cardiomyocyte injury in vitro

Following HR, a decrease in cardiomyocyte vitality and an increase in LDH levels in the culture serum were observed. To assess the protective effect of ulinastatin, cardiomyocytes were treated with ulinastatin prior to HR. Cell vitality was found to increase and LDH levels in the culture serum were found to decrease in the HR+ulinastatin group compared with the HR group (Fig. 1).

Figure 1 LDH levels in the cell culture serum and cardiomyocyte vitality measured using MTT assay (n=6). LDH levels were found to increase and cell vitality was observed to decrease following HR treatment. HR-induced injury was attenuated by ulinastatin compared with the HR group (n=5). (A) LDH levels in the cardiomyocyte culture serum. (*P<0.001 vs. Con and #P<0.001 vs. HR). (B) Cardiomyocyte vitality measured using MTT assay (*P<0.001, *P=0.005 vs. Con; #P=0.011 vs. HR). LDH, lactate dehydrogenase; Con, control; HR, hypoxia-reoxygenation.

Ulinastatin attenuates HR-induced cardiomyocyte autophagy in vitro

In order to analyze cardiomyocyte autophagy, LC3 protein expression was assessed using western blot analysis. The LC3-II/LC3-I expression ratio was used to measure the relative autophagy levels in cardiomyocytes. HR was found to induce cardiomyocyte autophagy, which was attenuated by ulinastatin treatment (Fig. 2).

Figure 2 LC3 protein expression detected using western blot analysis (n=5). (A) Western blot showing LC3 protein expression in the different groups. (B) Ratio of LC3-II/LC3-I expression in different groups. LC3-II was upregulated by HR and was attenuated by ulinastatin. *P<0.001, P=0.017 vs. Con and #P=0.008 vs. HR. LC3, light chain 3; Con, control; HR, hypoxia-reoxygenation.

Ulinastatin inhibits cardiomyocyte autophagy through mTOR in vitro

In order to investigate the mechanism underlying the anti-autophagic effect of ulinastatin in cardiomyocytes, the protein expression of p-Akt (Ser-473), p-mTOR (Ser-2448) and p-P70S6K (Thr-389) was analyzed. Treatment with ulinastatin prior to HR significantly increased the protein expression of p-Akt, p-mTOR and p-P70S6K compared with the HR group (Fig. 3). Furthermore, the LC3-II/I expression ratio was significantly increased in the cells treated with rapamycin+ulinastatin compared with those treated with ulinastatin, indicating that rapamycin attenutates the ulinastatin-induced inhibition of autophagy (Fig. 4).

Figure 3 p-Akt, p-mTOR and p-P70S6K protein expression detected using western blot analysis (n=5). (A) Western blot analysis of p-Akt, p-mTOR and p-P70S6K protein expression in the different groups. (B) Relative protein expression of p-Akt, p-mTOR and p-P70S6K in the different groups. p-Akt, p-mTOR and p-P70S6K expression were found to be downregulated by HR, and this HR-induced downregulation was attenuated by ulinastatin. (*P<0.001, P=0.017 vs. Con and #P=0.008 vs. HR). p-, phosphorylated; Akt, protein kinase B; mTOR, mammalian target of rapamycin; P70S6K, P70S6 kinase; Con, control; HR, hypoxia-reoxygenation.

Figure 4 Rapamycin regulates LC3 protein expression (n=4). (A) Western blot of LC3 protein expression from the different groups. (B) Ratio of LC3-II/LC3-I expression in the different groups. Ulinastatin-induced LC3-II downregulation was attenuated by rapamycin during HR in vitro. *P<0.001 vs. HR and #P=0.002 vs. ulinastatin. LC3, light chain 3; HR, hypoxia-reoxygenation.

Ulinastatin attenuates myocardial IR injury by inhibiting autophagy in vivo

To demonstrate the protective myocardial and anti-autophagic effects of ulinastatin, the rats were treated with ulinastatin (1×104 U/kg body weight) 30 min prior to IR. Ulinastatin inhibited IR-induced cardiomyocyte autophagy and reduced the relative area of the infarct, serum LDH levels and inflammation (Fig. 5).

Figure 5 Ulinastatin inhibits cardiomyocyte autophagy and attenuates IR-induced myocardial injury (n=6). (A) Mid-myocardial cross sections of triphenyltetrazolium chloride-stained hearts. Dark blue area, nonischemic zone; white area, INF; red area, AAR (B) AAR/LV and INF/AAR ratios. No significant difference was observed in AAR/LV among the Con, IR and ulinastatin groups. However ulinastatin was found to significantly attenuate the INF/AAR ratio in the ulinastatin+IR group compared with the IR group. *P<0.001 vs. Con and #P<0.001 vs. IR. (C) LC3-II/LC3-I ratio in the different groups. LC3-II was upregulated by IR and downregulated by ulinastatin in vivo. *P<0.001, *P=0.047 vs. Con and #P<0.001 vs. IR. (D) LDH levels in the blood serum. Ulinastatin reduced LDH levels in the blood serum compared with the IR group. *P<0.001, *P=0.002 vs. Con and #P<0.001 vs. IR. IR, ischemia reperfusion; AAR, area at risk; LV, left ventricle; Con, control; LC3, light chain 3; INF, infracted tissue.

Discussion

Ulinastatin has been proposed as a myocardial protective agent. Ulinastatin has been reported to improve myocardial contractility, reduce myocardial infarct size and decrease serum levels of creatine kinase and cardiac troponin I following myocardial IR injury in vivo (1). In the present study, cultured primary cardiomyocytes were treated with ulinastatin prior to HR in vitro. Ulinastatin was found to improve cell vitality and reduce LDH levels, thus ulinastatin may have a protective effect against HR injury in cardiomyocytes in vitro. Furthermore, the myocardial protective effect of ulinastatin was was found to be associated with various mechanisms in an in vivo rat heart model. It has previously been reported that ulinastatin may contribute to the recovery of cardiac function following reperfusion by reducing mitochondrial dysfunction and maintaining energy production (16). Moreover, the protective effect of ulinastatin may be associated with anti-inflammatory effects. For example, ulinastatin has been reported to reduce the expression of TNF and IL-6 and upregulate that of IL-10 and -13 (12,17–21).

It has been reported that TNF stimulates autophagy, while IL-13 suppresses autophagy through stimulating the phosphoinositide 3-kinase/mTOR signal transduction pathway (4,9,22). Therefore, ulinastatin may inhibit autophagy via the downregulation of TNF and the upregulation of IL-13. Autophagy is regulated by autophagy-related genes (Atgs), among which beclin 1 is required for the vesicle nucleation step of autophagy. Autophagosome formation involves two complexes, Atg12-Atg5-Atg16 and Atg4-Atg7-Atg3. These complexes are involved in the conversion of the soluble form of LC3 (LC3-I) to the autophagic vesicle-associated form (LC3-II), which is used as a marker of autophagy (23). The LC3-II/LC3-I ratio has been used for analyzing autophagy in numerous studies. In a previous study on an IR model, autophagy was found to be markedly enhanced and inhibition of autophagy, via the downregulation of beclin 1, was observed to have a protective effect in vivo (24).

In the present study, HR-induced LC3-II protein expression was found to be downregulated by ulinastatin in vitro. Furthermore, ulinastatin was observed to upregulate p-Akt, p-mTOR and p-P70S6K protein expression. To assess whether ulinastatin inhibits HR-induced cardiomyocyte autophagy through activating mTOR, cells were treated with the mTOR-specific inhibitor, rapamycin prior to HR and ulinastatin treatment. Rapamycin treatment was found to upregulate LC3-II, indicating that rapamycin attenuated the anti-autophagic effect of ulinastatin. Therefore, ulinastatin may protect cardiomyocytes against HR injury by inhibiting autophagy through activating Akt/mTOR. This may be associated with ulinastatin-induced anti-inflammatory effects.

In order to investigate the anti-autophagic effect of ulinastatin in vivo, a rat IR model was established. Certain rats were treated with ulinastatin (1×104U/kg body weight) 30 min prior to IR. Inflammation was found to be reduced by ulinastatin. Furthermore, myocardium LC3-II protein expression, myocardial infarct size and serum LDH levels were observed to be reduced in the ulinastatin group compared with the IR group.

In conclusion, the present study demonstrated that ulinastatin has an important protective role against IR injury by regulating autophagy via the mTOR signaling pathway. Furthermore, the protective effect of ulinastatin may be associated with its anti-inflammatory effect.

Acknowledgements

The present study was supported by the Tian Pu Research Foundation, Nature Science Foundation of Science and Technology Commission of Shanghai Municipality (grant no. 112ZR1454600) and the National Natural Science Foundation of China (grant no. 81200181).

References