Recombinant Human Cytoglobin In The Treatment Of Hyperlipidemia And Atherosclerosis In Rats And Its Mechanism

Hyperlipidemia refers to the abnormal fat metabolism making the plasma lipids existing in the vessel than normal.Hyperlipidemia is called dyslipidemia,which always shows that high blood cholesterol(TC)or triglyceride(TG)or low high-density lipoprotein cholesterol(HDL-C).Lipid is insoluble or sparingly soluble in water,walways accompanied by elevated serum lipoproteins.Clinical manifestations of hyperlipidemia is that lipid hich must be combined with a protein to form lipoproteins.So the high concentration of liqid is deposition in the dermis caused xanthomas and in the vascular endothelium caused slow progression of atherosclerosis.Atherosclerosis(AS)is an artery disease mainly involving the aorta,coronary arteries,cerebral arteries,renal arteries,lower extremity arteries,which is characterized by lipid accumulation on arterial intima,thrombosis,fibrosis and arterial media calcification.This disease will make the blood vascular wall thicken and stiff,blocked,and artery distal ischemia,these will lead to local tissue necrosis.AS is one of the most common diseases of the cardiovascular system,involving the body’s vital organs and blood vessels,resulting in serious consequences,such as coronary atherosclerosis can cause angina,arrhythmias;cerebral atherosclerosis can cause cerebral ischemia resulting in dizziness and headache;lower extremity atherosclerosis can lead to intermittent claudication.With the continuous improvement of social development and people’s living standards,AS caused the rapid increase of morbidity and mortality,which has become the leading cause of death worldwide.Therefore,atherosclerosis has become a medical research hotpot.One feature of AS is the foam cells which is the main component of lipid plaque.In recent years,a large number of studies have shown that oxidative modification of low density lipoprotein(ox-LDL)plays an important role on the formation of macrophage foam cells.Exposed to free radicals or other oxidizing agents,the unsaturated fatty acids which are rich in natural LDL core,with a series of reactions,become chemically modified LDL,including acetylated LDL,malondialdehyde(MDA),and ox-LDL.Chemical modification of LDL is identified and metabolized by the scavenger receptor,with no regard to intracellular cholesterol content,which makes a large number of chemically modified LDL accumulated in cells,lead to the formation of foam cells.And here we want get more study abut ox-LDL.Studies have shown that,ox-LDL promotes the development of AS in the following ways:(1)ox-LDL activates endothelial cells and stimulate endothelial cells to secrete chemokines,adhesion factor and colony stimulating factor,which makes monocytes attach to endothelium and move to subintimal proliferating and differentiating into macrophages.Macrophage changes into foam cells by the accumulation of ox-LDL using scavenger receptor.(2)Ox-LDL causes smooth muscle cell proliferating and migrating by inducing smooth muscle cells and macrophages secreting platelet-derived growth factor and basic fibroblast growth factor.(3)ox-LDL’s cytotoxic effects can directly damage endothelial cells,inhibit the synthesis of endothelium-derived relaxing factor,and destroy arterial wall’s normal systolic and diastolic function.Thus,inhibiting the vascular lipid peroxidation is an important means of mitigation and prevention of atherosclerosis.The current primary treatment of atherosclerosis is a healthy lifestyle,regular physical exercise and symptomatic treatment,such as lipid regulating drugs,simvastatin,fenofibrate;unsaturated fatty acids;antioxidants,vitamin C,vitamin E,β-carotene and probucol etc;endothelial cell protection drug,sulfated polysaccharide,alginate sodium carbonate;traditional Chinese medicine,cassia,hawthorn,Salvia.Since there is no effective treatment of AS,which has become a global health problem,it is an urgent need to develop a truly effective drug.CYGB is one member of a newly discovered class of six-coordinated heme-containing protein(hexacoordinate globin super-family,hxHb),which expressed in animals,bacteria,plants[1].CYGB’s crystal structure shows that it is like the other hxHb,whose ligand binding regulatory mechanism is completely different from the hemoglobin(Hb)and myoglobin(Mb),the classic five-coordinated blood red fibroin[2].The physiological functions of Hb and Mb,such as breathing oxygen-carrying capacity,have already been clearly defined,but the physiological function hxHb is not very clear.In mammals,CYGB is widely expressed in the fibroblasts and fibroblast-like cells of liver,heart,intestine,kidney,lung and pancreas.CYGB has the capacity of reversible binding with 02,NO and CO[2].CYGB selectively overexpressed in oxidative stress response area,such as the hippocampus,thalamus,hypothalamus[3].CYGB is presumed to participate in the transport,storage and sensing of oxygen,the production of collagen and the response of oxidative stress and cellular hypoxia.CYGB’s mRNA increases in cells under hypoxia and CYGB’s express level also increases after the activation of fibroblast in liver,pancreas,kidney[4].CYGB was increased by H2O2 specifically.When siRNA used to reduces CYGB’s gene expression levels,cell death rate increased inducing by oxidative stress[5].CYGB produced a lot when tissue damage and CYGB became an effective scavenger of reactive oxygen species[6].At the molecular level,CYGB may act as nitric oxide dual oxidase,removing excess NO[7].NO produces by nitric oxide synthase and nitrite mobilization.NO has a very important role in the vasculature:NO,which as a second messenger regulating the vascular endothelial vasomotor function,inhibit LDL oxidation.But when the macrophages engulf a lot of ox-LDL,they will produce large amounts of intracellular oxidation products,such as ROS,NO,MDA,resulting in cell damage and inducing apoptosis of foam cells[8].In vivo and in vitro experiments showed that overexpressed recombinant human cell globin(rhCYGB)protects hepatic stellate cells from oxidative stress and inhibits their differentiation into fibroblast-like cells[9].Similarly,oxidative stress is the main reason for the arterial endothelial injury and,consequentially,the atherosclerosis.We then hypothesized that the oxygen-carrying rhCYGB can regulate NO levels and antioxidant capacity,and thus benefiting the treatment of atherosclerosis.In this study,there is containing three experimental sections.The first section was focused on testing the efficacy of the rhCYGB treatment in hyperlipidemia.We use the engineering bacteria PET28a-rhCygb-BL21(DE3)to get rhCYGB.Firstly,planted the bacteria on Kan+ LB plates and picked one single colony,proliferation culture and ultrasonic cytolysis.And protein renaturation and preliminary purification was accomplished by molecular sieve chromatography and then further purified by affinity chromatography.Finally protein concentration was determined using the BCA protein assay kit.We ran SDS-PAGE protein electrophoresis gel electrophoresis of rhCYGB and analyzed rhCYGB’s purity calculations on gray BandScan 5.0 software.We used rhCYGB T-AOC kit to detect purified antioxidant biological activity.The purity of affinity chromatography purified rhCYGB is about 93%,and the antioxidant activity of about 95U/mg.50 male SD rats were randomly divided into 2 groups in the first part of experiment:10 rats in normal control group and40 rats in experiment group.The rats in normal control group were fed with normal diet and those in the experiment group were fed with high-fat diet.After four weeks,10 rats of experiment randomly selected for serum liquid detection,and the result confirmed the model work well.40 rats were randomly divided into groups of 3mg/kg rhCYGB treatment group(n=8),6mg/kg rhCYGB treatment group(n=8),9mg/kg rhCYGB treatment group(n=8),propylene glycol alginate sodium in the treatment group(n=8),high-fat group(n=8).Those rats got eight weeks of treatment by subcutaneous injection,once every 2 days.Normal control group fed normal diet during the experiment,while rhCYGB treatment group,sodium alginate ester treatment group and model group fed high-fat diet throughout the experiment.After the treatment finished,all the rats were taken aortic blood and tissue samples after fasting and ether anesthesia.HepG2 were cultured in 6-well plates with 10%fetal bovine serum in DMEM medium,37 ℃,5%CO2.Cells were counted and adjusted number to 1×106/ml,and then divided into groups:normal control group;Oleic acid treatment groups;0.6mmol/L Oleic acid treated group;Oleic acid +rhCYGB group:0.6mmol/L Oleic acid plus various doses of rhCYGB(5μg/mL,10 μg/mL,15 μg/mL)treatment;simvastatin treatment group:0.6mmol/L Oleic acid plus 1μg/mL propylene glycol alginate sodium.All groups were treated for 72h and got test.The cells were collected to do MTT assay,measure triglyceride content by high performance liquid chromatography HPLC,measure intracellular glutathione peroxidase GPx,malondialdehyde(MDA)and superoxide enzyme(SOD)by kit.Our results show that TG,TC and LDLC were noticeably higher in the treatment group using 9mg/kg rhCYGB than in the control group but much lower than those in the high-fat diet group.The HDLC in the 9mg/kg rhCYGB treatment group was significantly higher than the control and high-fat groups.The results of HepG2 cells show that,rhCYGB has no toxic effects on cells in the dose 30μg/mL.15μg/mL rhCYGB reduced intracellular total triglyceride content and the level of intracellular MDA,increased SOD,GPx activity.As we know,hyperlipidemia continued progress will lead to the accumulation of lipids in the artery wall,causing arterial damage and atherosclerosis formation.The initial study of rhCYGB effect on hyperlipidemia provides a guideline in arterial atherosclerosis research later.The second section was focused on testing the efficacy of the rhCYGB treatment in atherosclerosis in rats.41 male SD rats were randomly divided into 2 groups in the first part of experiment:eight rats in normal control group and 33 rats in experiment group.The rats in normal control group were fed with normal diet and those in the experiment group were fed with high-fat diet.And the experiment group were got intraperitoneal injection of vitamin D3,a total of 700 000 IU/Kg,4 times,once/every 3 days,based on the high-fat diet.After four weeks,one rat of experiment randomly selected for HE staining pathological section,and the result confirmed the model work well.32 rats were randomly divided into groups of 4mg/kg rhCYGB treatment group(n=8),7mg/kg rhCYGB treatment group(n=8),propylene glycol alginate sodium in the treatment group(n=8),high-fat group(n=8).Those rats got eight weeks of treatment by subcutaneous injection,once every 2 days.Normal control group fed normal diet during the experiment,while rhCYGB treatment group,sodium alginate ester treatment group and model group fed high-fat diet throughout the experiment.After the treatment finished,all the rats were taken aortic blood and tissue samples after fasting and ether anesthesia.Our results show that TG,TC,LDLC,and Ca+ were noticeably higher in the treatment group using 7mg/kg rhCYGB than in the control group but much lower than those in the high-fat diet group.The HDLC in the 7mg/kg rhCYGB treatment group was significantly higher than the control and high-fat groups.The vessel wall intima of 7mg/kg rhCYGB treatment group was a little bit worse that the control group,but the tunica media was intact and non-calcified,which is much better than the high-fat diet group.So AS situation in this treatment group was greatly improved in comparison with the high-fat group.Furthermore,the 7mg/kg rhCYGB treatment group show significantly lower intima-media thickness ratio comparing to high fat diet group.The third part of the experiment:we used human acute leukemia monocytic line THP-1 to study the mechanism in vitro.It was focused on exploring the possible mechanisms of atherosclerotic arterial therapy using rhCYGB.This was mainly done by demonstrating whether rhCYGB would suppress the foam cells formation from the macrophages,which ox-LDL-induced from the monocytic cell line THP-1.THP-1 were cultured in 6-well plates with 10%fetal bovine serum in RPMI 1640 medium,37 ℃,5%CO2.Cells were counted and adjusted number to 1×106/ml,then added phorbol ester(PMA)in the cell suspension to a final concentration of 50ng/ml,transferred to six-well plates,each well was added 2mL,stationary culture 72h.THP-1 cells were induced to differentiate into macrophages,and then divided into groups:normal control group;ox-LDL treatment groups:50μg/mL of ox-LDL-treated group;ox-LDL + rhCYGB group:50μg/ox-LDL plus various doses of rhCYGB(5 μg/mL,10 μg/mL,15 μg/mL)treatment;simvastatin treatment group:50μg/mL of ox-LDL plus μg/mL simvastatin.All groups were treated for 24 h and get test.The cells were collected to do MTT assay,measure intracellular cholesterol content by high performance liquid chromatography HPLC,measure intracellular glutathione peroxidase GPx,malondialdehyde(MDA),superoxide enzyme(SOD),and NADPH oxidase by kit.And the cells were tested the fluorescent intensity of ROS and NO by fluorescent probe.The results show that,rhCYGB has no toxic effects on cells in the dose 30μg/mL.15μg/mL rhCYGB reduced intracellular total cholesterol content and the ratio of cholesterol ester to total cholesterol,which is helpful to reverse the formation of foam cells.RhCYGB can reduce the level of intracellular MDA,inhibit NADPH oxidase activit,decreased intracellular ROS,NO concentration,increased SOD,GPx activity.Both the three parts of experimental results show that rhCYGB can improve and reverse atherosclerosis in rats,whose curative effect is better than simvastatin.In vitro cell experiments,rhCYGB can inhibit ox-LDL damaging macrophages,lower the intracellular lipid,resist to oxidative stress and reverse foam cell formation.CYGB is a protective protein,widely expressed in various organs of the body.Its expression increased to defense adverse factors in ischemia,oxidative stress,fibrosis and other stress conditions.Since the low expression in our body,we try to make CYGB into genetically engineered drugs used for biological research or clinical use,exploring its therapeutic efficacy and mechanism of action.