Mechanism of Algal Blue Protein in Reducing Myocardial Ischemia-Reperfusion Injury in Rats

Abstract: Objective: To observe the effects of phycocyanin on myocardial ischemia-reperfusion injury in rats and analyze the possible mechanisms. Methods Thirty-six rats were selected and divided into blank control group, model group and treatment group by random number table method. The model and treatment groups were modeled by repeating silk ligation 3 times and reperfusion of the anterior descending branch of the left coronary artery for 45 min. After modeling, the treatment group was treated with 10% alginate suspension at 200 mg/(kg - d) by gavage for 7 days, and the blank control group and the model group were treated with an equal volume of saline by gavage. Rat coronary artery cell cycle protein-dependent kinase inhibitor p27 and p21 were detected by image analysis; inducible nitric oxide synthase (iNOS) was detected by immunohistochemical staining; and intracardiac artery serum superoxide dismutase (SOD), creatine kinase isoform (CK-MB), malondialdehyde (MDA), and nitric oxide (NO) were detected by enzyme-linked immunosorbent assay. ).

 

Results: In the treatment group, the rat coronary artery cell cycle protein-dependent kinase inhibitor p27 and p21 were highly expressed, iNOS-positive cells and the levels of SOD and NO were elevated, and the levels of CK-MB and MDA were significantly reduced, and the differences were statistically significant when compared with that of the model group (P＜0.05). Conclusion Alginin has the effect of inhibiting myocardial ischemia-reperfusion injury.

 

The main cause of myocardial ischemia-reperfusion injury is not ischemia per se, but rather the massive production of oxygen free radicals after re-flow, and these lipid peroxidation products often cause irreversible damage to cardiomyocytes, which is also the main cause of cardiac mortality [1-2]. It has been shown that the coronary cell cycle protein-dependent kinase inhibitors p27 and p21 play a key role in the regulation of ischemic myocardial tissue remodeling [3-4].

 

In this process, nitric oxide (NO) is also involved in the migration, proliferation and neovascularization of vascular endothelial cells[5] . As the NO rate-limiting enzyme, changes in the activity of inducible nitric oxide synthase (iNOS) will affect NO production and indirectly regulate vascular endothelial function[6] . Thus, any factors affecting cell cycle protein-dependent kinase inhibitors p27, p21, and iNOS can improve or worsen myocardial ischemia-reperfusion injury. In view of this, our group selected Spirulina extract phycocyanin, which has the ability to inhibit lipid peroxidation and antioxidant activity, as a research target and observed its effects on myocardial ischemia-reperfusion injury in rats.

 

1 Materials and Methods 1 .1 Materials

1 .1 .1 Animals and groupings  

Thirty-six male rats, SPF grade, 5 weeks old, weighing 150.0 ± 10.5 kg were selected. The rats were purchased from Wuhan Jintangxin Breeding Technology Co., Ltd [Certificate of Conformity: SCXK (E) 2014-0015] and kept in the SPF laboratory of Hubei Medical College [Certificate of Conformity: SYXK (E) 2018-0031]. The rats were divided into blank control group, model group and treatment group, each with 12 rats after being numbered by random number table before modeling. The experiments were conducted in accordance with the 3R principle to provide animals with humane care and fully satisfy their welfare: during the experiments, the animals were allowed to eat and drink freely, and the temperature of the incubation room was controlled at 22-26 ℃, the relative humidity was controlled at 50%-75%, and the animals were exposed to light and darkness alternately every 12 hours.

 

1 .1 .2 Major equipment and reagents  

BW-BM1103 small animal respirator and integrated informatization signal acquisition and processing system were purchased from Chengdu Taimeng Software Co. Mouse anti-p27 and p21 polyclonal antibodies were purchased from Wuhan Three Eagles Biotechnology Company Limited (Lot No. LS-C211591 and LS-C211597, respectively). iNOS, NO, superoxide dismutase (SOD), creatine kinase isoform (CK-MB) and malondialdehyde (MDA) test kits were purchased from Nanjing Jianjian Institute of Biological Engineering (Lot No. LZ087), (batch numbers: LZ087, H1098, H1012, H1054, H1104).

 

1 .2 Experimental Methods

1 .2 .1 Modeling and Treatment  

The model and treatment groups were anesthetized with 3% sodium pentobarbital 30 mg/kg intraperitoneally. After the rats were anesthetized, they were fixed on the operating table and connected to the BW-BM1103 small animal ventilator and the BL-420S Biofunctional Experiment Recording System (to record the Ⅱ-lead electrocardiograms), and then underwent open-heart surgery. After the open-heart surgery, the anterior descending branches of the left coronary artery were located, and the left ventricle was blocked with a silk thread to cause acute ischemia, 45 min later, the silk thread was released and reperfused for 45 min, which was repeated three times. After 45 min, the wire was loosened and reperfused for 45 min, and this procedure was repeated for three times, and the successful replication of the model was based on the appearance of presystole, T-wave elevation, and ST-segment elevation or upward shift in the Ⅱ-lead electrocardiogram after reperfusion [7]. After the model was completed, the chest cavity was closed, sutured in layers, sterilized, and the animals were kept separately. The blank control group was not anesthetized and did not undergo the above procedures. Immediately after modeling, the treatment group was treated with 10% alginate suspension at 200 mg/(kg - d) by gavage for 7 days, while the blank control group and the model group were treated with equal volume of saline by gavage.

 

1 .2 .2 Detection indicators and methods  

Coronary artery cell cycle protein-dependent kinase inhibitor p27 and p21 were detected by image analysis; iNOS expression and the number of positive cells were detected by immunohistochemical staining; and the levels of SOD, CK-MB, MDA and NO were detected by enzyme-linked immunosorbent assay.

1.3 Statistical processing The data were analyzed using SPSS21.0 statistical analysis software. Measurement data were expressed as x ± s. Comparison of group means was performed by one-way analysis of variance (ANOVA), and comparison of group means was performed by two-sample paired t-test, with P < 0.05 indicating that the differences were statistically significant.

 

2 Results

2 .1 Cell cycle protein-dependent kinase inhibitory factors in rats of all groups

Comparison of the expression levels of p27 and p21 The rats in the model group expressed low levels of cell cycle protein-dependent kinase inhibitory factors p27 and p21, and the differences were statistically significant compared with those in the blank control group (P<0.05). The rats in the treatment group expressed high levels of cell cycle protein-dependent kinase inhibitory factors p27 and p21, and the differences were statistically significant (P<0.05) when compared with the model group. The differences were statistically significant (P < 0.05) compared with the model group.

 

2.2 Comparison of iNOS and NO levels in rats of each group  

The levels of iNOS and NO in the model group were lower than those in the blank control group, and the differences were statistically significant (P<0.05). The levels of iNOS and NO in the treatment group were significantly higher than those in the model group, and the differences were statistically significant (P < 0.05). See Table 2.

 

2.3 Comparison of myocardial enzyme indexes of rats in each group SOD level of rats in the model group was lower than that of the blank control group, while CK-MB and MDA levels were significantly higher than that of the blank control group, and the differences were statistically significant (P< 0.05); SOD level of rats in the treatment group was higher than that of the model group, while CK-MB and MDA levels were significantly lower than that of the model group, and the differences were statistically significant.

(P < 0 .05). See Table 3 .

 

3 Discussion

In the research of myocardial ischemia-reperfusion injury, in addition to targeted basic research on pathophysiology and physiology, medical practitioners should also devote themselves to searching for or screening highly effective and low-toxicity drugs for the treatment or prevention of ischemia-reperfusion injury. Phycocyanin, a photosynthetic pigment protein extracted from Spirulina, was first found to have many pharmacological effects in animal experiments, such as antioxidant, reducing oxidative stress, maintaining homeostasis, detoxification, anti-inflammatory, anti-allergic, anti-tumor and so on[8-10] . In the present study, based on its ability to scavenge hydroxyl radicals and hydroperoxyl radicals, we analyzed its mechanism of action to reduce the effects of myocardial reperfusion injury by observing the factors that can reflect the remodeling of myocardial tissue in myocardial ischemia-reperfusion injury.

 

The results of this study showed that coronary artery p27 and p21 were highly expressed in the treatment group, and the levels of SOD, iNOS, and NO were significantly higher than those in the model group, and the levels of CK-MB and MDA were significantly lower than those in the model group. It has been shown that cell cycle protein-dependent kinase inhibitor p21 and p27 can regulate the proliferation cycle of myocardial and vascular smooth muscle cells, and the high expression of cell cycle protein-dependent kinase inhibitor p27 and p21 can promote the proliferation of myocardial and vascular smooth muscle, thus affecting the reconstruction of damaged myocardial tissues after myocardial ischemia/reperfusion.11-12 NO is involved in this process, and NO is also involved in the reconstruction of damaged myocardial tissues after myocardial ischemia/reperfusion. In this process, NO is involved in the migration and proliferation of vascular endothelial cells, and is an important initiator of myocardial tissue remodeling after ischemia-reperfusion[13] .

 

Under normal physiological conditions, NO and iNOS remain in homeostasis, but when myocardium suffers ischemia-reperfusion injury, iNOS activation is increased, and its high expression induces NO synthesis, which plays a role in regulating vascular endothelial function to promote the remodeling of damaged myocardial tissues[14-15] . From the results of the present study, phycocyanin acts on this link and promotes the expression of cyclin-dependent kinase inhibitors p27 and p21 and iNOS in ischemia-reperfusion myocardial cells, and this effect directly induces NO synthesis, which regulates vascular endothelial function and plays a key role in ischemia-reperfusion myocardial tissue remodeling. In addition, this study also observed that alginate has the effect of scavenging free radicals and inhibiting lipid peroxidation, which is reflected in the increase of SOD activity in the treatment group and the decrease of the levels of CK-MB, a product of cell death, and MDA, a product of lipid peroxidation, in the ischemia-reperfusion group compared with that in the model group.

 

In conclusion, the mechanism by which phycocyanin reduces myocardial ischemia-reperfusion injury in rats may be related to its ability to promote the expression of cell cycle protein-dependent kinase inhibitors p27 and p21 and to enhance the synthesis of iNOS-induced NO, which can regulate the function of the vascular endothelium, promote the remodeling of damaged myocardial tissues, and alleviate the destruction of myocardial cells by lipid peroxidation products to mitigate the effects of myocardial ischemia-reperfusion injury.

 

References:

[1 ] Feng Shuo, Zhang Ruiyan . Factors influencing the occurrence of left ventricular remodeling after acute myocardial infarction[J ] . International Journal of Cardiovascular Disease, 2019 , 46(5 ): 267-269 .

[2 ] WU Xiaoyan, MIAO Lin, ZHENG Rui, et al. Research progress of myocardial ischemia-reperfusion injury [J ] . Chinese Journal of Clinical Pharmacology, 2016 , 12(11):1043-1045 .

[3 ] Cao Fai, Xu Liqin, Liu Qingzhong, et al. Protective effect of phycocyanin on acute myocardial ischemia-reperfusion injury in rats[J ] . China Medicine Herald, 2017 , 14(14):25-28 .

[4 ] LIU CZ , ZHENG HZ , XIE L , et al . Decreased miR-208 induced is chemia myocardial and reperfusion injury by targeting p21 [J ] . Pharm azie , 2016 , 71(12):719-723 .

[5 ] NAGASAKA Y , FERNANDEZ BO , GARCIA-SAURA MF , et al .Brief periods of nitric oxide inhalation protect against myocar - dial ischemia-reperfusion injury [ J ] .Anesthesiology , 2008 , 109 (4): 675-682 .

[6 ] LU Shenlu, WANG Ruiying, NIU Yaqiong, et al. Study on the trend of serum nitric oxide level in intermittent hypoxia mice[J ] . China Cardiovascular Disease Research, 2019 , 17(11):1028-1031 .

[7 ] WANG Wenjie, CHEN Hongying, PENG Jixia, et al. Effect of Rhodiola rosea glycosides on the expression of vascular endothelial growth factor after preconditioning for myocardial ischemia in rats [J ] . China Medical Journal, 2015 , 12(9 ): 29-33 .

[8 ] WU Xianjun, YANG Hong, SHENG Yi, et al . Cloning, expression and antioxidant activity of recombinant proteins of three cyanobacterial phycocyanin β-subunit deacylated protein genes [J ] . Jiangsu Journal of Agriculture, 2018 , 34(5):998-1004 .

[9 ] Liu Huihui, Jiang Liangqian, Wang Yujuan, et al. Current status of the application of algal blue protein against tumor[J ] . Tumor Control Research, 2018 , 45(6):420-424 .

[10] LIU Qi, LI Wenjun, TANG Zhihong, et al. Research progress on the role of algal blue protein in the prevention and treatment of oxidative stress-related diseases[J ] . Marine Science, 2017 , 41(10):132-138 .

[11 ] LI H , ZOU T , MENG S , et al . p21 protects cardio myocytes a- gainst ischemia-reperfusion injury by inhibiting oxidative stress [J ] .Mol Med Rep , 2018 . 17(3):4665-4671 .

[12] CHU Xian-Ming, LI Bing, DU Ri-Ying, et al. Effect of P27 on luminal stenosis after balloon injury in rat thoracic aorta[J ] . Chinese Journal of Arteriosclerosis, 2006(5):417-421 .

[13] YANG Shasha, ZHOU Li . Effect of alginate on myocardial ischemia-reperfusion injury in rabbits[J ] . Journal of Changchun University of Traditional Chinese Medicine, 2017 , 33(6):868-870 .

[14] ZHANG Wei, ZHAO Lijun, LI De'an, et al. Pigment epithelium-derived factor is involved in arsenic-induced vascular endothelial cell dysfunction[J ] . Chinese Journal of Endemic Diseases, 2019(2):107-110 .

[15] LIU Zhaoya, XU Xi, TANG Yixin, et al. Research progress on the regulation of endothelial-type nitric oxide synthase activity and cardiovascular diseases[J ] . Journal of Central South University (Medical Edition), 2016 , 41(61 ): 632