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The roles of CD59 gene in anti-atherosclerotic effect of C-phycocyanin

- May 11, 2018 -

The roles of CD59 gene in anti-atherosclerotic effect of C-phycocyanin


XU Yingjie1, LI Bing1,*, WANG Peilin1,*, CHU Xianming2, YANG Fan1

1. Department of Biology, Medical College of Qingdao University, Qingdao 266021, China;

2. Department of Cardiology, the Affiliated Hici Hospital of Qingdao University Medical College, Qingdao 266000, China



To evaluate the effects of CD59 gene in occurrence and development of atherosclerosis with presence of C-phycocyanin (CPC), 40 mice with ApoE gene deletion (ApoE-/-) recruited were randomly divided into four groups: control group, C-phycocyanin treated group, CD59 treated group and C-phycocyanin(CPC)+CD59 treated group. All mice fed on high-fat diet, and were treated with drug intervention respectively. At the end of the 12th week, CD59 mRNA level was determined by RT-PCR, and CD59 protein expression was detected by Western blot. Blood fat levels of mice were also determined, and aortic roots were taken out for observing the degree of atherosclerotic plaques formation. We found that both C-phycocyanin and CD59 gene could lower blood fat level and inhibit atherosclerosis formation, and the combination of CD59 gene and C-phycocyanin could more effective. In addition, C-phycocyanin could promote the CD59 mRNA and protein expression, thus further inhibit the progress of atherosclerosis, which might be the mechanism of antiatherosclerosis roles of C-phycocyanin.

Key words:  Atherosclerosis; CD59; C-phycocyanin; ApoE-/-



Atherosclerosis (AS) is a systemic disease. Hyperlipidemia, hypertension, smoking, secondary hyperlipidemia, genetic factors, and other factors all contribute to the development of AS. At present, the search for economical, safe, and effective anti-atherosclerotic drugs has become a major hotspot and problem in today's medicine [1]. Atherosclerosis is a chronic inflammatory and immunological disease. Immune and non-immune factors cause endothelial dysfunction, which initiates the development of atherosclerosis. Complement is an important effector in the immune system, and there is abundant evidence that the complement system is involved in the development of atherosclerosis [2]. Studies have shown that the activation products of the complement system, such as regulatory proteins and complement receptors, can be detected in atherosclerotic lesions, especially in vulnerable plaques and ruptured plaques, and have been found through studies of human lesions. There was a positive correlation between the amount of deposition of the membrane attack complex and the extent of lesion progression. The complement regulatory protein CD59 is a key regulator of complement membrane attack complex (MAC) assembly and has the role of inhibiting complement membrane attack complex formation [3]. Liposomes are circular closed vesicles composed of lipid bilayer molecules. As non-viral vectors for gene transfer, they have the advantages of being non-toxic, easy to use, non-immunogenic, and high transfection rate [4]. The target gene has a pharmacokinetic effect after being injected through the tail vein [5]. C-phycocyanin (CPC) in Spirulina platensis has high medical value, such as improving blood lipid metabolism, anti-oxidation, anti-tumor, anti-aging, and improving immunity.[6] Because of its safe, non-toxic, high water solubility, high content, easy extraction and other characteristics are widely used in cosmetics and food additives. Atherosclerosis is caused by endothelial dysfunction, and phycocyanin can promote animal cell regeneration to repair damaged endothelial cells and induce necrotic cell apoptosis, which in turn regulates endothelial function in the body. And it can effectively suppress the occurrence of hyperlipidemia by improving blood lipid metabolism and scavenging the physiological activities of free radicals and other foreign substances. It is speculated that phycocyanin has a positive effect on the treatment of atherosclerosis. This article will combine the complement regulatory protein CD59 with the characteristics of CPC, by high-fat diet ApoE-/- mice transfected with CD59 gene, CPC gavage, and the combined effect of CD59 gene and CPC, to study CPC on arterial porridge the effect of CD59 expression in sclerosclerotic mice and whether the effect of CPC on antiatherosclerosis in ApoE-/- mice is to slow down or suppress the development of atherosclerosis by promoting the expression of CD59 gene, to show the Mechanism of phycocyanin in anti-atherosclerotic.


1. Materials and methods

Experimental materials and instruments

40 ApoE-/- mice, male and female in equal proportions, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Spirulina platensis purchased from Ocean University of China. Phycocyanin may directly purchased from Zhejiang Binmei Biotechnology Co.,Ltd. CD59 gene high expression plasmid CD59pIRES was provided by the Department of Immunology, Qingdao University Medical School. Lipofectamine 2000 transfection reagent was purchased from Invitrogen. Cholesterol was purchased from Sigma.Pig bile was purchased from Solarbio. Anti-mouse CD59 polyclonal antibody was purchased from Santa Corporation, USA The EndoFree Plasmid ezFlow Maxiprep Kit was purchased from BIOMIGA, and the RNAiso PLUS kit and PrimeScript RT-PCR Kit were purchased from TaKaRa. Nucleic acid protein analyzers were purchased from Eppendorf. desktop thermostatic oscillators were purchased from the U.S. Biochemical Instrument Factory in Taicang. cryogenic high-speed centrifuges KR-1500 were purchased from KUBOUA. and electronic balances and particle ice machines were purchased from Sanyo Corporation of Japan.


1.2 Extraction of phycocyanin

250 g Spirulina platensis was fully ground to a powder and added to a concentration of 0.1 mmol/L , pH 7.0, 500 ml phosphate buffer (PBS).  Soaked at 4 °C for 30 min, and mixed well. Freeze and thawed 10 times at -20 °C and 38 °C, stand for 30 min, centrifuge at 4 000 r/min for 40 min, mix the supernatant with an equal volume of saturated ammonium sulfate solution, and salt out at 4 °C for 24 h. The phycocyanin was obtained by centrifugation at 4000 r/min for 40 minutes after salting out and was dissolved in 50 ml PBS and placed in a dialysis bag with a relative molecular mass of 8 000 to 14 000. The ammonium ion was removed by dialyzing in 1 000 ml of PBS. CPC suspension [6]. The nucleic acid protein analyzer measures the concentration of the CPC solution.

Phycocyanin may directly purchased from Zhejiang Binmei Biotechnology Co.,Ltd.


1.3 Modeling and administration methods

1.3.1 Modeling methods and doses 

40 ApoE-/- mice were randomly divided into 4 groups, 10 in each group, male and female, and fed with high-fat diet (formulation: ordinary diet 80%, 15% pig oil, 2% cholesterol, 1.5% whole milk powder, 1.3% egg yolk powder, 0.2% pig bile salt) [7]. Dosage: The mass concentration of CPC is 31.32 mg/ml, administered at 100 mg/kg by gavage, 0.1 ml per mouse (about 30 g); the mass concentration of CD59pIRES plasmid is 117 μg/ml, After being embedded in liposomes, a CD59-pIRES-liposome complex was formed at a mass concentration of 97.5 μg/ml, and each mouse was injected with approximately 0.5 ml.


1.3.2 Grouping Method Group 

Group 1 : In the control group, only the high-fat diet was fed as a control. Group 2: the CPC treatment group was administered by intragastric administration with a CPC suspension twice a week.  Group 3: In the CD59 treatment group, the CD59 pIRES-liposome complex was administered via tail vein injection to achieve the purpose of transfecting the CD59 gene into mouse cells [8] twice per week. 

Group 4: In the CD59+CPC-treated group, CD59-pIRES-liposome complexes were injected through the tail vein of mice at the same time as intragastric administration of CPC, twice a week. Four groups of mice were fed high-fat diet and treated with drugs for 12 weeks.

1.4 Detection of CD59 mRNA and protein expression in mice of each group

1.4.1 Detection of CD59 mRNA levels in blood by RT-PCR The mice were given an empty stomach for 12 h. After anesthesia, the eyeballs were collected and blood was taken. Total RNA from whole blood of 4 groups of mice was extracted using RNAiso PLUS kit. Reverse transcription reactions were performed using the PrimeScript RT-PCR Kit kit to synthesize cDNA. Then take the reverse transcription product for PCR amplification. According to the primer design software design. CD59 upstream primer: 5'-TGGACAATCACAATGGGAA TC-3'; CD59 downstream primer: 5'-TGCTGCCAGAAATGGA GTCAC-3'. The PCR reaction was performed on the PCR instrument under the following conditions, predenatured at 94 °C for 2 min, denatured at 94 °C for 30 s, annealed at 47 °C for 1 min, and extended at 68 °C for 2 min. After 40 cycles of steps 2 to 4, the final elongation at 68 °C was obtained. 7 min; the length of the amplified product was 378 bp, and the length of the internal reference GAPDH amplified product was 509 bp.


1.4.2 Western blot detection of CD59 protein expression in tissue cells Dissection of 4 groups of mice, liver, spleen, heart and aorta and other tissues were removed, placed in pre-cooled physiological saline, rinsed several times to wash away bloodstains. In each group, weigh 0.45 g of the tissue into a mortar and grind the tissue according to the net mass of the tissue: the volume of the lysate is 1:2 and grind until the tissue is ground into a slurry. The protein was collected by centrifugation and the protein concentration was determined. Proteins were separated by SDSPAGE electrophoresis. The target proteins were transferred to PVDF membrane and blocked for 2 h. Rabbit anti-mouse CD59 (Mr 20 000) and rabbit anti-human internal standard β-actin (Mr 43 000) were added to the target antibody and incubated overnight at 4 °C. The horseradish peroxidase-labeled secondary antibody was incubated at room temperature for 2 h and chemiluminescence developed. The image was analyzed by Image J image analysis software [9].


1.5 Determination of biochemical levels of blood lipids

The rest of the blood is left to stand and examined for various biochemical indicators in serum: total cholesterol (TC), triglyceride (TG), apolipoprotein B (ApoB), and low-density lipoprotein (LDL) High-density lipoprotein (HDL) [10].


1.6 Atherosclerotic plaques were observed by HE staining.

The thorax was opened and the aortic roots were isolated to avoid crushing. Aortic specimens were fixed with 4% paraformaldehyde, and serial paraffin sections were made and stained with HE [11]. Atherosclerotic plaque formation was observed under a light microscope.


1.7 Statistical Analysis

SPSS17.0 statistical software was used to analyze and process the data. The results were expressed as the mean±standard deviation. The comparison between groups was performed using the t-test. P<0.05 was considered a significant difference in statistics. 


2.1 CD59 mRNA expression RT-PCR results of ApoE-/- mice in each group were analyzed by gel image analysis system. The expression of CD59 mRNA in each group was significantly different (Fig. 1a). According to the gray value analysis, compared with the control group, the CD59 mRNA expression level in the CPC treatment group increased, indicating that phycocyanin can promote the expression of CD59 mRNA in blood cells. The level of CD59 mRNA in the CD59 transfected group was also significantly higher than that of the control group, indicating that the CD59 gene has been successfully transfected into blood cells (Fig. 1b).

Fig 1 Determination of CD59 mRAN expression (C-Phycocyanin).jpg 



2.2 Expression levels of CD59 protein of ApoE-/- mice in each group

The expression of CD59 protein in mouse tissues was detected by Western blot. The results are shown in Figure 2a. Proteins with different expression levels of CD59 were detected in each group. Bands. The gray value of each group of protein bands was corrected and analyzed (Fig. 2b). Compared with the control group, the expression of CD59 protein in the CD59-treated group, the CPC-treated group, and the CPC+CD59-treated group was significantly increased. The difference in CD59 expression between the CPC+CD59-treated group and the control group was even more significant. The results show that CPC can promote the expression of CD59 protein in mouse tissues. The transfection of CD59 high expression plasmid also increased the expression of CD59 protein to some extent, indicating that the CD59 gene has been successfully transfected into mouse tissue cells. The above results are consistent with the detection results of mRNA levels.



Fig 2 Determination of CD59 protein expression in different groups (C-Phycocyanin).jpg 



2.3  Changes in serum lipid biochemical levels of ApoE-/- mice in each group

Compared with the serum lipid biochemical parameters of mice in the same-age high-fat diet control group and normal diet mice (Table 1), it was found that the high-fat diet control group was small The levels of TG, TC, ApoB, and LDL in mice increased significantly while HDL levels decreased, and the difference was significant (P<0.05). It has been proved that ApoE-/- mice develop hyperlipidemia after being fed with high-fat diet and can induce atherosclerosis. The serum lipid levels of ApoE-/- mice in each experimental group were compared and it was found that there was no significant difference in ApoB concentrations between the groups compared with the control group. The concentration of TG, TC, and LDL in the treatment group and the CD59 treatment group were significantly decreased (P<0.05), but the HDL concentration was significantly higher than that of the control group. Compared with the control group, the CD59+CPC treatment group had a more pronounced regulation of blood lipids (P<0.01, Table 2). The results showed that the blood lipid levels in the CPC-treated group and the CD59-treated group were significantly lower, and the CD59+CPC-treated group was more effective than the two alone, and could further reduce the blood lipid levels in the mice and slow down the hyperlipidemia. The formation of symptoms, thereby inhibiting the formation of atherosclerotic plaque.


Tab 1 Comparison of blood fat levels in normal diet mice and high fat diet control mice (C-phycocyanin).jpg 


Tab 2 Comparison of blood fat levels in the different groups of mice (C-phycocyanin).jpg 


2.4 Observation of atherosclerotic plaques in ApoE-/- mice in each group.

After arterial specimens were stained with HE and observed under microscope, significant morphological differences were observed in each group [12]. In the high-fat diet control group, the intima was thickened, the integrity was destroyed, and plaques formed. The smooth muscle cells of the middle layer migrated into the intima through the elastic plate, and they proliferated and transformed to form plaques (Fig. 3a). Compared with the control group, both the CPC-treated group and the CD59-treated group showed significant thickening of the intima and the destruction of the integrity, but no obvious atherosclerotic plaque formation was observed (Figure 3b, c). On the inside of the lumen of the CD59+CPC-treated group, there was a continuous and smooth layer of endothelial cells with no obvious thickening of the intima and no obvious plaque (Figure 3d). The results show that both CPC and CD59 can slow the formation of atherosclerotic plaque to a certain extent, and the combined effect of CD59 and CPC is more effective than the two alone, which can further slow down or even inhibit atherosclerotic plaque. Formation.


Fig 3 Cross sections of the aorta root from mice of different groups (C-phycocyanin).jpg 


3. Discussion

Atherosclerosis (AS) is a systemic disease. Hyperlipidemia, hypertension, smoking, secondary hyperlipidemia, genetic factors and other factors all contribute to the development of AS. A large number of epidemiological studies have demonstrated that hyperlipidemia is a risk factor for the development of atherosclerosis, and hyperlipidemia refers to an abnormal increase in plasma total cholesterol and triglycerides [1]. The severity of AS increases with an increase in plasma cholesterol levels. Oxidized LDL (ox-LDL) is currently considered to be the most important atherogenic factor and a major cause of damage to endothelial cells and smooth muscle cells. HDL can remove cholesterol from the arterial wall through the reverse cholesterol transport mechanism, and antioxidants can prevent the oxidation of LDL, and can inhibit the binding of LDL to receptors on endothelial cells to reduce their uptake [13]. Therefore, LDL concentration is the best indicator for determining AS, and HDL levels will decrease as AS lesions worsen. In this experiment, the TG, TC, and LDL concentrations of ApoE-/- mice fed a high-fat diet for 12 weeks were significantly higher than those of normal-fed ApoE-/- mice, and the HDL decreased, indicating the formation of hyperlipidemia (Table 1). Sections of the aortic roots under light microscopy revealed thickening of the intima, destruction of integrity, and obvious atherosclerotic plaque formation. The above results indicate that the atherosclerotic mouse model was successfully constructed.


The complement system is involved in the entire process of atherosclerosis. The amount of deposition of the membrane attack complex is positively correlated with the degree of progression of the lesion. Cell of differentiation 59 (CD59) is a glycosylphosphatidylinositol (GPI) anchor glycoprotein that is a key regulator of the complement membrane attack complex (MAC) assembly and inhibits complement membrane attack. The role of complex formation. CD59 binds to C8 and C9 and blocks the formation of MAC C5b-9 complex. It has the function of regulating complement activity and protecting the host cell from complement melting [14]. Foreign studies have found that CD59 is associated with trauma, inflammation, and the development of certain tumors [15]. In this experiment, RT-PCR and Western blot reactions confirmed that CPC can promote the expression of CD59 mRNA and CD59 protein in mice. In addition, intravenous liposome complexes are an effective form of gene transfection and can increase bioavailability [16]. In this experiment, the CD59 expression levels in the control and CD59-treated groups were compared (Figure 1, Figure 2). It was confirmed that the CD59 gene was successfully transfected into mouse blood and tissue cells by liposome-embedded mouse tail vein injection.


We further investigated the effects of phycocyanin and CD59 gene on the occurrence and development of atherosclerosis in ApoE mice. By serum biochemical markers and HE staining of aortic specimens, compared with the control group, both CPC intervention and CD59 transfection can reduce blood lipid levels to some extent (Table 2), inhibit intimal hyperplasia and atherosclerotic plaques. Block formation (Fig. 3b, c, d) slows the development of atherosclerosis, and the combined effect of CPC and CD59 is more effective, which can further slow down or even inhibit the formation of atherosclerotic plaque.


In summary, we found that phycocyanin can promote the expression of CD59 gene in atherosclerotic mice, and CD59 has been proved to have the function of lowering blood lipid level and delaying the development of atherosclerosis. Then we presumed that phycocyanin Antiatherosclerosis may be achieved by regulating the expression of the CD59 gene in ApoE-/- mice. This study provides new ideas and new methods for the treatment of atherosclerosis drugs and genes.



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