Cardioprotective effect of the polysaccharide from Ophiopogon japonicus on isoproterenol-induced myocardial ischemia in rats

Journal Pre-proof Cardioprotective effect of the polysaccharide from Ophiopogon japonicus on isoproterenol-induced myocardial ischemia in rats

Sairong Fan, Junfeng Zhang, Qi Xiao, Peng Liu, Yining Zhang, Enze Yao, Xiaoming Chen PII:

S0141-8130(19)40508-4

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.068

Reference:

BIOMAC 14372

To appear in:

International Journal of Biological Macromolecules

Received date:

20 December 2019

Revised date:

2 January 2020

Accepted date:

6 January 2020

Please cite this article as: S. Fan, J. Zhang, Q. Xiao, et al., Cardioprotective effect of the polysaccharide from Ophiopogon japonicus on isoproterenol-induced myocardial ischemia in rats, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.01.068

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© 2020 Published by Elsevier.

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Cardioprotective effect of the polysaccharide from Ophiopogon japonicus on isoproterenol-induced myocardial ischemia in rats Sairong Fana,b,1, Junfeng Zhanga,b,1,2, Qi Xiao a,b, Peng Liua,b, Yining Zhanga,b, Enze Yaoa,b, Xiaoming Chena,b,*

( a Institute of Glycobiological Engineering/School of Laboratory Medicine and Life

Zhejiang Provincial Key Laboratory of Medical Genetics, Key Laboratory of

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b

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Sciences, Wenzhou Medical University, Wenzhou 325035, China

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Laboratory Medicine, Ministry of Education, China, School of Laboratory Medicine

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Xiaoming Chen

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*Corresponding author:

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and Life Sciences, Wenzhou Medical University, Wenzhou, China 325035)

Sciences

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Institute of Glycobiological Engineering/School of Laboratory Medicine and Life

Wenzhou Medical University Wenzhou, 325035

Tel: 086-577-86699651 Fax: 086-577-86689717 E-mail: [email protected] Note:

1

These authors contributed equally to this work.

2

Present address: The children's hospital, Zhejiang university school of

medicine, Hangzhou, China. 1

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Abstract

The polysaccharide (OJP1), extracted from the root of Ophiopogon japonicus, is a well known traditional Chinese medicine used to treat cardiovascular diseases. The present study was set up to investigate the cardioprotective effect of OJP1 on isoproterenol (ISO)-induced myocardial ischemia injury in rats. Results showed that

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pretreatment with OJP1 (100, 200 and 300 mg/kg) significantly reduced ISO-induced

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ST-segment elevation and the heart index, attenuated the levels of marker enzymes

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(AST, LDH, CK and CK-MB), along with a significantly enhanced the activities of

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ATPases. Moreover, pretreatment with OJP1 not only enhanced the activities of SOD,

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GPx and CAT in serum and myocardium, but also decreased the level of MDA. The biochemical and histopathological analysis also showed that OJP1 can alleviate the

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myocardial injury induced by ISO. Taken together, our results indicated that oral

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administration of OJP1 offered significant cardioprotective effect against the damage induced by ISO through enhancement of endogenous antioxidants. Keywords:

Ophiopogon

japonicus

polysaccharides;

myocardial

ischemia;

Cardioprotetive; Antioxidant

1. Introduction

Cardiovascular disease (CVD) is the first cause of deaths and its incidence is rapidly increasing in worldwide, especially in developing countries [1,2]. Myocardial ischemia, which can initially present no symptoms but subsequently lead to serious 2

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clinical consequences, has become a common life-threatening disease causing many adverse effects on the heart [3,4]. Reperfusion therapy can rescue myocardial ischemia and myocardial stunning, however, clinical and animal studies have found that with reperfusion in the myocardial tissue, which can lead to damaged myocardia and newly forming myocardial injuries caused by reoxygenation still induce heart

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failure [5-7]. Therefore, prevention and treatment of ischemia/reperfusion-induced myocardial injury, thereby alleviating or preventing the occurrence of various heart

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diseases, has been a hot topic in treating ischemic heart disease [8].

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Numerous experimental evidences have suggested that traditional herbal medicines

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moieties exert more effective in the treatment of cardiovascular diseases, including

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ischemic heart disease [9,10]. Accumulated evidence has demonstrated that

[11,12].

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polysaccharides from medicinal plants have significant cardioprotective effects

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Ophiopogon japonicus (Thunb.) Ker-Gawl, is a well known traditional Chinese medicine used to treat cardiovascular and chronic inflammatory diseases for thousands of years, and has been confirmed in various experiments as having anti-ischemic, anti-arrhythmia, anti-inflammatory and microcirculation improvement, etc. [13]. Reports have showed that the polysaccharides isolated from O. japonicus have various biological activities, such as immunostimulation, anti-ischaemia, inhibiting platelets aggregation, and hypoglycemic [14,15]. In our previous study, we isolated and characterized a water-soluble polysaccharide (OJP1) from the roots of O. japonicus. OJP1 is a heteropolysaccharide with an average molecular weight of 35.2 3

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kDa and consisting of arabinose, glucose and galactose in a relative molar ratio of 1:16:8 [14]. OJP1 has been demonstrated with an anti-diabetic effect and improving cardiovascular performance against the injurious effects of diabetes [16]. However, the effects of the polysaccharide (OJP1) in rats with isoproterenol (ISO)-induced ischemic injury have not been reported yet.

administration

of

OJP1

could

protect

the

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Therefore, the present study was conducted to investigate whether the oral heart

against

ISO-induced

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ischemia/reperfusion-induced myocardial injury in rats by studying the serum marker

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enzymes and antioxidant parameters, and then explore the underlying mechanisms of

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2. Materials and methods

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cardioprotective effect and the health benefits of the polysaccharide.

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2.1. Materials and Chemicals

The roots of O. japonicus were collected in Zhejiang province (China). The polysaccharide (OJP1) was isolated from the roots of O. japonicus, and purified by DEAE-52

cellulose

and

Sephadex

G-100

as

described

previously

[14].

DEAE-cellulose and Sephadex G-100 were obtained from Sigma. Isoproterenol (ISO) and Inderal were purchased from Shanghai Harvest Pharmaceutical Co. Ltd, Shanghai, China. The assay kits for aspartate transaminase (AST), lactate dehydrogenase (LDH), Nitric Oxide Synthase (NOS), glutathione peroxidase (GPx)，creatine kinase (CK), and creatine phosphokinase-MB (CK-MB) were purchased from Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China. The assay kits for nitric 4

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oxide (NO), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), Na+-K+-ATPase and Ca2+-Mg2+-ATPase were purchased from Beyotime Institute of Biotechnology, Shanghai, China. The assay kits for endothelin-1 (ET-1) and ischemia modified albumina (IMA) were purchased from Westang Biotechnology Co., LTD, Shanghai, China. Immunohistochemistry kits were purchased from Zhongshan All the other chemicals

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Goldenbridge Biotechnology Co., LTD., Beijing, China.

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used were of analytical grade.

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2.2. Experimental animals

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Sprague-Dawley rats (body weight 200 ± 20 g) were purchased from Shanghai

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Laboratory Animal Center, Shanghai, China. The animals were acclimatized at least one week before starting the experiment. Before and during the experiment, the rats

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were housed under controlled environmental conditions of temperature (22 ± 2 °C) in a 12 h light and dark cycle, and maintained on standard food pellets and tap water ad

experiments.

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libitum [16]. This study was approved by the College committee for animal

The animals (used in this experiment) handling procedures were performed in strict accordance with the P. R. China legislation the use and care of laboratory animals, with the guidelines established by Institute for Experimental Animals of Wenzhou Medical University.

2.3. Experimental design

After one week of acclimatization, a total of 42 rats were randomly divided into six 5

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groups with seven animals in each group and subjected to respective treatment as follows: Group normal control (NC): the animals were received only saline for 14 days. Group ISO control (MC): the animals were received saline for 14 days and administered with ISO (4 mg/kg, intraperitoneally) for 2 days on the 13th and 14th day.

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Group OJP1-100 (OJP1-100): the animals were treated with 100 mg/kg/d of OJP1 for 14 days and administered with ISO (4 mg/kg, intraperitoneally) for 2 days on the

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13th and 14th day.

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Group OJP1-200 (OJP1-200): the animals were treated with 200 mg/kg/d of OJP1

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for 14 days and administered with ISO (4 mg/kg, intraperitoneally) for 2 days on the

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13th and 14th day.

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Group OJP1-300 (OJP1-300): the animals were treated with 300 mg/kg/d of OJP1 for 14 days and administered with ISO (4 mg/kg, intraperitoneally) for 2 days on the

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13th and 14th day.

Group Inderal (Inderal): the animals were treated with 10 mg/kg of Inderal for 14 days and administered with ISO (4 mg/kg, intraperitoneally) for 2 days on the 13th and 14th day.

At the end of the experiment (15th day, after 24 hour inject ISO last time), the rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). Electrocardiograph (ECG) was recorded continually, and ST-segment elevation or depression (expressed in mv) in normal and experiment animals were considered.

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After recording the ECG, blood was collected in polystyrene tubes without the anticoagulant. Serum was immediately separated by centrifugation at 3000 rpm at room temperature for 10 min. Samples were stored at -70 °C for further biochemical assay.

Animals were sacrificed by cervical dislocation. The heart, kidney and liver were

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removed quickly, blotted dry and weighed.

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2.4. Biochemical assay in the serum

The activities of AST, LDH, CK and CK-MB were measured in serum using

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respective commercial detecting kits following manufacture’s protocol.

The concentrations of NO, NOS, ET-1, and IMA were measured in serum through

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enzymatic methods using the automated biochemistry analyzer (ARCHITECT C8000,

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USA). Assays were performed using commercially available kits following the manufacture’s instructions.

The activity of SOD, GPx and CAT, as well as the concentration of MDA were measured in serum using respective commercial available kits following manufacture’s protocol. In brief, the activity of SOD was measured by the production of O2- anions. The activity of GPx was estimated by the analysis of GSH in the enzymatic reaction. The activity of CAT was estimated by the absorbance of N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinonemonoimine,

which

was

a

red-colored oxidation product. The concentration of MDA was spectrophotometrically 7

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measured by the absorbance of a red-colored product with thiobarbituric acid. 2.5. Myocardial antioxidant activity assays

Heart was homogenized (1:10 w/v) in cold 0.9% saline solution by using a motor-driven Teflon glass homogenizer, followed by centrifugation at 3500 rpm for 10 min at 4 °C, then the supernatant was collected for estimation of various

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biochemical parameters, such as myocardial SOD, GPx, CAT and MDA.

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2.6. Myocardial Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities assay

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The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in the heart were

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determined spectrophotometriclly using respective commercial diagnostics kits following manufacture’s instructions.

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2.7. Histopathological observation of rat heart

At the end of the experiment, hearts from all the groups were subjected to histopathological studies. Processing of tissue samples followed established procedures

[16]. Briefly,

the heart

tissues

were

fixed in

10% neutral

paraformaldehyde, embedded in paraffin wax. Paraffin section (5 μm) were cut on glass slides, deparaffinized by xylene, ethanol, 95% ethanol, 80% ethanol sequentially, and stained with hematoxylin and eosin (H&E). After that, the samples were redeparaffinized by 80% ethanol, 95% ethanol, ethanol, xylene sequentially, and then were sealed with neutral resin, histological examinations were carried out by optical microscopy, and then photomicrographs were taken. 8

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2.8. Statistical analysis of the data

All results were presented as mean ± S.D. Data were analyzed by one-way analysis of variance (ANOVA), followed by Student’s t-test. P values less than 0.05 were considered significant.

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3. Results and discussion

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3.1. Effects of OJP1 on heart, kidney and liver index in ISO-induced rats

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Isoproterenol, a -adrenergic agonist, has been known to exert both inotropic and

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chronotropic cardiac effects, and induces myocardial ischemia by increasing the force

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and frequency of myocardial contractions [17]. To investigate the protective effect of OJP1 on myocardial ischemia, an animal model induced by ISO was used in the study.

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As shown in Table 1, the kidney or liver indices of ISO-administered rats (MC group)

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had no significant difference as compared to those of normal control rats (NC group). However, the heart index of ISO-administered rats (MC group) was higher than that of normal control rats (NC group), indicating increase in size of the heart. Rats pre-treated with OJP1 (100, 200 or 300 mg/kg) showed a significant (P < 0.01) reduction in the heart index as compared to ISO-administered group, although it did not act in a dose-dependent manner (Table 1). Inderal pre-treated also significantly decreased the heart index of the animals, when compared with that of ISO-administered animals (P < 0.01). Patel et al. have reported that the heart weight increment in ISO-induced rats might be due to the increased water content, 9

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oedematous intramuscular space and related to the onset of myocardial necrosis [18]. Pretreatment of OJP1 significantly decreased the heart weight in ISO-induced rats, which indicated the protective effect of OJP1 on ISO-induced myocardial injury in rats.

Table 1 Effects of OJP1 on heart, kidney and liver index in myocardial ischemic rats a Liver index (mg/g)

NC

2.44±0.04

31.27±1.71

8.05±0.15

MC

3.74±0.05b

31.72±1.64

7.33±0.08

OJP1-100

3.06±0.19c

30.89±0.97

7.59±0.30

OJP1-200

2.92±0.10c

30.93±1.59

8.11±1.13

OJP1-300

3.23±0.11c

Inderal

3.09±0.15c

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Kidney index (mg/g)

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a

Heart index (mg/g)

7.32±0.59

32.83±3.18

8.56±0.28

b

P < 0.01 (compared with NC group).

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Data represent mean ± S.D. (n = 7 for each group). c P < 0.01 (compared with MC group).

30.30±2.53

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3.2. Effects of OJP1 on electrocardiograph parameters in ISO-induced rats

Electrocardiograph (ECG)-abnormalities are the main criteria generally used for the definite diagnosis of myocardial ischemia and infarction. Myocardial ischemia disturbs the cells’ membrane potential, which engenders electrical potential difference. ST-segment elevation reflects the potential difference in the boundary between ischemic and normal myocardium.

[18,19]. As shown in Fig. 1, normal rats showed

a normal electrocardiograph pattern. ISO-alone administered rats showed a marked elevation in ST-segments. Pretreatment with OJP1 (100, 200 and 300 mg/kg, respectively) or Inderal (10 mg/kg) in ISO-administered rats showed a significant (P 10

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< 0.01) decrease in ST-segment when compared with ISO-alone administered rats (Fig. 1). The ST-segment elevation induced by ISO might be due to the consecutive loss of cell membrane in injured myocardium, and pretreatment with OJP1 markedly reduced ST-segment depression confirmed the cardioprotective effect of OJP1 in

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ISO-induced rat.

Fig. 1. Representative electrocardiogram tracings and ST-segments changes of control and experimental animals. A-F: normal control group (NC), ISO-treated alone administered group (MC), ISO with 100 mg/kg OJP1 pretreated group (OJP1-100), ISO with 200 mg/kg OJP1 pretreated group (OJP1-200), ISO with 300 mg/kg OJP1 pretreated group (OJP1-300), ISO with 10 mg/kg Inderal pretreated group (Inderal, positive control) respectively. G: ST-segments changes. Data are presented as mean  SD (n = 7).

##

P < 0.01 (Compared with NC group),

group). 11

**

P < 0.01 (Compared with MC

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3.3. Histopathological examination of myocardial tissues

To further investigate the protective effect of OJP1 on the myocardial tissues, the histopathology of rat’s heart was examined. As shown in Fig. 2, photomicrograph of normal control group revealed a normal myofibrillar architecture with striations, branched appearance and arranged neatly, and the morphology of myocardial cells

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was uniform (Fig. 2A). However, ISO-alone administered rats (MC group) showed

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widespread myocardial structure disorder, marked myofibrillar degeneration and

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confluent necrosis of cardiac muscle fibres leading to impairment of membrane

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structural and functional integrity (Fig. 2B), which displayed that the myocardium

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was injured by ISO-treated. Pretreatment with OJP1 (100, 200 and 300 mg/kg, respectively), the tissue sections showed some infiltration with neutrophil

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granulocytes, interstitial edema and some discontinuity with adjacent myofibrils, but

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the morphology of cardiac muscle fibres was relatively well preserved with no evidence of necrosis and less cellular infiltration when compared to MC group (Fig. 2C, 2D and 2E), indicated that OJP1 has significant cardioprotective effects. Photomicrograph of Inderal pre-treated group also showed well preserved myocardial structure with less cellular infiltration and necrosis (Fig. 2F).

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Fig. 2. Histological evaluation of myocardial tissue. A-F: normal control group (NC),

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ISO-treated alone administered group (MC), ISO with 100 mg/kg OJP1 pretreated

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group (OJP1-100), ISO with 200 mg/kg OJP1 pretreated group (OJP1-200), ISO with

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300 mg/kg OJP1 pretreated group (OJP1-300), ISO with 10 mg/kg Inderal pretreated

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group (Inderal, positive control) respectively (magnification,  20). 3.4. Effects of OJP1 on serum marker enzymes in ISO-induced rats

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The heart, one of the most active organs, contains a large number of enzymes. Among then, cytosolic enzymes namely AST, LDH, CK, and CK-MB serve as the diagnostic markers of myocardial tissue damage. Increased activities of these cellular enzymes in the serum reflect the cellular damage and loss of functional integrity and/ or permeability of cell membrane [20]. To further determine the cardioprotective effect of OJP1 in ISO-administered rats, we characterized the activities of serum biomarkers, which are released into the blood during the occurrence of myocardial ischemic reperfusion (IR) and commonly used to reflect the severity of cardiomyopathy [21,22]. As shown in Fig. 3, the activities of diagnostic marker 13

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enzymes (AST, LDH, CK and CK-MB) in the serum of ISO-alone administered rats (MC group) were significantly (P < 0.01) higher than those of normal control animals (NC group). The increased activities of AST, LDH, CK and CK-MB in serum of ISO-induced rats may due to the leakage of these enzymes from the heart as a result of myocardial necrosis induced by ISO. However, pretreatment with OJP1 (100, 200

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and 300 mg/kg, respectively) or Inderal (10 mg/kg) showed a significant (P < 0.01) reduction in the activities of all serum diagnostic marker enzymes compared to MC

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group, which means OJP1 can maintain the membrane integrity and permeability,

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thereby limiting the leakage of these enzymes, indicating that OJP1 attenuates

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ISO-induced tissue injuries.

Fig. 3. Effects of OJP1 on serum marker enzymes activities in control and ISO-induced rats. (A) The activity expressions of AST, (B) The activity expressions of LDH, (C) The activity expressions of CK, (D) The activity expressions of CK-MB. Data are presented as mean  SD (n = 7). ##P < 0.01 (Compared with NC group), **P < 0.01 (Compared with MC group). 14

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3.5. Effects of OJP1 on NO, NOS, ET-1 and IMA in ISO-induced rats To elucidate the effect of OJP1 on myocardial ischemic reperfusion injury in ISO-administered rats, the important factors (NO, ET-1 and IMA) in serum were investigated. As shown in Fig. 4, the level of NO was significantly (P < 0.01) decreased in ISO-alone administered rats (MC group), and the activity of NOS was

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also significantly (P < 0.01) reduced, compared with normal control animals. Pretreatment with OJP1 (100, 200 and 300 mg/kg, respectively), the levels of NO and

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activities of NOS were significantly (P < 0.01) increased, compared with those in MC

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group. The level of ET-1 was significantly (P < 0.01) increased in MC group,

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compared with normal control group (Fig. 4). However, the levels of ET-1 were

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significantly (P < 0.05 or P < 0.01) reduced in all OJP1 pretreatment groups when

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compared with that in MC group. NO, a key signaling messenger in the cardiovascular system, is important to preserve normal vascular physiology, and

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decreased NO bioavailability is proposed as one of the central factors common to cardiovascular disease [23,24]. ET-1 is the predominant isoform expressed in vasculature and the most potent vasoconstrictor currently known, and over-expressed of ET-1 will contributes to high blood pressure and heart disease [25]. The interplay of NO and ET-1 has a great relevance in the physiological regulation of blood pressure and vascular tone [16]. In the present study, the delicate balance between vasoconstriction and vasodilation might be broke by ISO-induced the decreased NO level and increased ET-1 level in rats. Whereas pretreatment of OJP1 significantly decreased the ET-1 level and increased the NO levels seems to rebuild the balance 15

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between vasodilation and vasoconstriction to protective the cardiovascular physiology in ISO-induced rats. Ischemia modified albumin, which is generated immediately following myocardial ischemia, is a novel marker of for ischemic injury and oxidative damage [26]. The level of IMA were significantly (P < 0.01) increased in MC group, compared with

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normal control group (Fig. 4). However, the levels of IMA were significantly (P <

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indicating the cardioprotective effect of OJP1.

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0.01) reduced in all OJP1 pretreatment groups when compared with MC group,

Fig. 4. Effects of OJP1 on the levels of NO, NOS, ET-1 and IMA in control and ISO-induced rats. (A) The levels of NO, (B) The activity expressions of NOS, (C) The levels of ET-1, (D) The activity expressions of IMA. Data are presented as mean  SD (n = 7). ##P < 0.01 (Compared with NC group), *P < 0.05 and **P < 0.01 (Compared with MC group). 16

Journal Pre-proof 3.6. Effects of OJP1 on myocardial Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities in ISO-induced rats

During ischemia, anaerobic myocardial metabolism predominates, reducing the amount of ATP produced [4], which may induce myocardial damage including ischemic cardiac dysfunction, etc. [27]. Fig. 5 showed the effects of OJP1

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pretreatment on myocardium Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities in

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experimental rats. Myocardial Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities were

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significantly (P < 0.01) decreased in ISO-alone administered rats (MC group),

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compared with those in the normal control animals. Pretreatment with OJP1 at doses

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of 100, 200, or 300 mg/kg obviously increased myocardial Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities in rats versus those of the MC group, suggesting that

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oral administration of OJP1 prevented ISO-induced cardiac dysfunction possibly via

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enhancement of myocardial Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities. This was further supported by the decreased levels of cardiac marker enzymes (AST, LDH, CK and CK-MB), indicating pretreatment with OJP1 attenuates the myocardium tissue damage in ISO-induced rats.

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Fig. 5. Effects of OJP1 on myocardial Na+-K+-ATPase and Ca2+-Mg2+-ATPase

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activities in ISO-induced rats. Data are presented as mean  SD (n = 7). #P < 0.05

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(Compared with NC group), *P < 0.05 (Compared with MC group).

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3.7. Effects of OJP1 on serum and myocardial antioxidant activity in ISO-induced rats

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Oxygen-derived free radicals, which can damage various biological targets, are

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closely related with the cardiac injury [27]. To further elucidate the protective effect of OJP1 on myocardial ischemic injury in ISO-administered rats, the effect of OJP1

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on the antioxidant activity was investigated. As shown in Table 2, the activities of SOD, GPx and CAT were significantly (P < 0.01) decreased in both serum and heart tissues of the ISO-alone administered rats (MC group), along with an increase of MDA, compared with those in the normal control rats (P < 0.05 or P < 0.01). However, the serum or heart tissues from pretreatment with OJP1 (100, 200, and 300 mg/kg) groups showed a significant (P < 0.05 or P < 0.01) increase the activities of endogenous antioxidants (SOD, GPx, and CAT), but a significant decrease in MDA level (P < 0.05 or P < 0.01), compared with those in MC group. These data demonstrated for the first time that administration of OJP1 could effectively fight 18

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against the oxidative stress associated with ischemic injury in ISO-induced rats. Oxidative stress is well documented role in the pathophysiological mechanism of a wide variety of clinical disorders including ischemic heart disease as well as ISO-induced myocardial ischemic injury [28,29]. Free radical scavenging enzymes such as SOD, GPx and CAT are the first line of the antioxidant defense system,

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eliminating reactive oxygen radical such as superoxide anion, hydrogen peroxide, and hydroxyl radical, and help to protect the tissue against oxidative damage [19]. SOD is

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usually found in the plasma membrane, and reduction in the SOD level is also a

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prominent marker of oxidative stress. CAT, a tetrameric hemoprotein, acts as a

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catalyst for the removal of hydrogen peroxide [30]. GPx offers protection to the

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cellular membranes from the peroxidative damage by reducing hydrogen peroxide and

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lipid peroxides [31]. In this study, a significantly lower activity of these enzymes was observed in both serum and myocardium of ISO-induced rats when compared to

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normal control rats. Suppression of these enzymes leads to the accumulation of these oxidants and make myocardial cell membranes more susceptible to oxidative injury. Pretreatment with OJP1 significantly increase the activities of all the above enzymes in both serum and mycocardium in ISO-induced rats, suggesting the antioxidant activity of OJP1 on ISO-induced rats. MDA, a major end production of lipid peroxidation, is generated under high levels of un-scavenged free radicals and affects the fluidity and permeability of cell membrane and ultimately leads to changes in cell structure and function, and is regarded as an index of cellular damage and cytotoxicity [22]. A significant increase 19

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in the levels of MDA in both serum and myocardium of ISO-induced rats was observed when compared to normal control rats. On the contrary, Pretreatment with OJP1 could decrease the MDA content elevation in both serum and myocardium when compared to MC group. The decreased level of MDA in serum and myocardium might be due to the enhanced activities in antioxidant enzymes (SOD, GPx and CAT).

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It was quite possible that the free radicals induced by ISO were effectively neutralized or scavenged. These findings suggest that OJP1 could considerably improve cellular

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antioxidative defense against oxidative stress, which is attributed to the therapeutic

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nature of OJP1 against peroxidative injury. These results indicated that OJP1 offered

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significant protection against the ISO-induced myocardial injury in rats through

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enhancement of endogenous antioxidants activity.

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Journal Pre-proof Table 2 Effects of OJP1 on activities of SOD, GPx, CAT and levels of MDA in myocardial ischemic rats a NC

MC

OJP1-100

OJP1-200

OJP1-300

Inderal

SOD (U/mL)

285.29±16.18

197.51±15.73c

273.44±12.16e

227.40±8.16d

246.38±16.54 d

298.75±22.22 e

GPx (U/mL)

2295.81±127.08

1728.14±151.03c

2252.69±113.56e

2004.79±135.05

2227.54±145.54 d

2389.22±114.24 e

CAT (U/mL)

1.90±0.08

1.16±0.03c

1.85±0.05e

1.25±0.03d

1.49±0.05 e

2.02±0.07 e

MDA (nmol/mL)

3.57±0.11

6.18±0.22c

3.75±0.16e

3.95±0.17 e

3.77±0.19 e

SOD (U/mg protein)

287.02±42.37

184.17±25.64c

278.36±34.89e

243.04±12.56 d

242.74±25.55 d

GPx (U/mg protein)

479.07±49.05

343.11±19.84b

426.92±36.80d

390.12±14.17d

385.57±22.36 d

417.41±18.45 e

CAT (U/mg protein)

2.79±0.55

1.01±0.25c

3.60±0.25e

2.79±0.39e

2.33±0.55 d

2.96±0.07 e

MDA (nmol/mg

5.62±0.66

9.85±1.00c

5.15±0.76e

5.70±1.17e

7.07±0.91 d

5.60±0.97 e

re

-p

242.52±22.84d

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protein)

3.84±0.21e

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Heart

of

Serum

Data represent mean ± S.D. (n = 7 for each group). b P < 0.05, c P < 0.01 (compared with NC group). d P < 0.05, e P<0.01 (compared with MC group).

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4. Conclusions

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a

In summary, the present study demonstrated for the first time that OJP1 exerts significant cardiopretective effects against ISO-induced myocardial ischemic injury in rats. The myocardial protective effect could be associated with enhancement of antioxidant enzyme activities and improved cardiovascular performance against the injurious. These findings might be rational to understand the beneficial effects of OJP1 on cardiovascular diseases, and will be helpful to develop novel drugs and functional foods in ischemic heart disease.

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Declaration of competing interest

The authors declare no competing financial interests.

Acknowledgments

This work was supported by Key Discipline of Zhejiang Province in Medical

of

Technology (First Class, Category A).

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Author statement: Sairong Fan: Validation, Conceptualization, Writing-Original draft preparation, Writing-Reviewing and Editing. Junfeng Zhang: Conceptualization, Investigation, Formal analysis. Qi Xiao: Investigation. Peng Liu: Investigation. Yining Zhang: Investigation. Enze Yao: Investigation.

Xiaoming Chen: Supervision,

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Conceptualization, Writing-Reviewing and Editing.

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Journal Pre-proof Highlights

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► OJP1 significantly decreased the ST-segment elevation induced by ISO. ► OJP1 significantly attenuated the levels of myocardial marker enzymes induced by ISO. ► OJP1 significantly increased serum and myocardial antioxidant enzyme activities. ► OJP1 has cardioprotective effect on ISO-induced ischemic reperfusion injury in rats.

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