CN101019848B - Application of ferricitras in preparation of medicine to prevent and treat angiosteosis - Google Patents
Application of ferricitras in preparation of medicine to prevent and treat angiosteosis Download PDFInfo
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Abstract
The present invention relates to the application of ferric citrate in preparing medicine for preventing and treating calcification of blood vessel. The present invention has determined curative effect. Experiment proves that ferric citrate can intervene calcification of blood vessel and inhibit calcification effectively, and may reverse calcification of blood vessel.
Description
Technical field
The present invention relates to the angiosteosis field, is the purposes of ferric citrate in the treatment of vascular calcification, especially relates to the research field of control cardiovascular heterotopic calcification, specifically, is the application of ferric citrate in control cardiovascular heterotopic calcification.
Background technology
Angiosteosis (also satisfactory vascular system heterotopic calcification) is and the closely-related a kind of pathological change of clinical cardiovascular disease, mainly betide arterial blood tube wall and cardiac valve, closely related with aging, atherosclerotic vascular lesion, aortic stenosis and diabetes, end stagerenaldisease etc., can increase acute myocardial infarction, peripheral blood vessel ischemic episode and postangioplasty inner membrance and the generation of serious clinical events such as tear, be cardiovascular disease incidence rate and predict mortality factor.
Angiosteosis mainly occurs in two position points of blood vessel wall: inner membrance and middle film.Calcification betides inner membrance and is called inner membrance calcification (inner membrance calcification), mainly is associated with atherosclerosis; Film calcification (middle film calcification) during film was called during calcification betided, it is independent of the existence of coronary atherosclerosis pathological changes, mainly betides pathological states such as aging, diabetes, uremia.The calcification in two sites has different morphology and pathological characteristics, sees table.The inner membrance calcification betides the lipid striped and forms the phase, and form is the hydroxyapatite crystal calcium deposition that distribution is filled the air in little being, and matrix vesicle in formation and the ossified similar cell of physiological.Middle film calcification betides in the environment of no inflammatory cell infiltration and lipidosis, and macrophage and mastocyte are not found in film calcification site in people's carotid artery, and the elastic layer of film is found matrix vesicle in the human artery, and the mechanism of middle film calcification is unclear at present.
The contrast of table inner membrance calcification and middle film calcification
| The inner membrance calcification | Middle film calcification | |
| Morphology | Diffusivity calcium deposition | Continuous wire calcium deposition |
| Linked groups is learned | Inflammatory cell (macrophage mastocyte) lipid | Smooth muscle cell; Elasticin |
| Relevant disease | Atherosclerosis | Diabetes; Chronic renal disease; Monckeberg ' s sclerosis |
The cardiovascular system heterotopic calcification is special, initiatively, an adjustable process.Hydroxyapatite, matrix vesicle, type i collagen and non-collagen synosteosis albumen (NCPs) that the method discovery of application SABC and in situ hybridization occurs in the physiological ossific process comprise that osteopontin, substrate Gla albumen, Bone Gla protein, osteonectin and bone morphogenetic protein (BMPs) etc. are present in the tremulous pulse and the valve of atheromatous plaque, calcification; The birth of MGP knock out mice 2 months endogenous cause of ill aortic valve, elastic force and flesh large artery trunks generation extensive calcification are died from arteriorrhexis and heart failure; The clone of CVCs cell discloses the cell that has similar parietal cell or mesenchymal cell in the blood vessel wall, can spontaneous formation calcification brief summary, and GF-β
1, 25-oxycholesterol, 17 beta estradiols and 1, the 25-vitamin D
3Deng the bovine aortic smooth muscle cells calcification that can promote In vitro culture.Angiosteosis and physiological sclerotin form similar, are initiatively, adjustable process.
The very complicated process that the cardiovascular system calcification is a multi-pathogenesis, multipath, participated in by multiple molecule, its key link be target cell the multiple factor of startup effect and inside and outside of various extraneous factors (as aging, lipidosis, atherosis, necrosis etc.) (as 1, the 25-vitamin D
3, TGF-β
1Deng) facilitation under, gene phenotype changes, gradually to osteoblast differentiation, secretion osteoblast sample substrate, and excretory bone matrix can act on cell, both interact, and finally form calcification.
Though modern medicine study has realized that the harm of cardiovascular system heterotopic calcification, still there is not effective Therapeutic Method at present.The calcium channel blocker Therapeutic Method of at present relatively approving in the industry and adopting, zoopery shows to be effective aspect the minimizing arterial calcium deposition, but the therapeutic response in different angiosteosis zones to calcium channel blocker is different, and clinical effectiveness is also not satisfied on the whole.Have the women who takes Hormone Replacement Therapy after the report menopause lower, but estrogen can not be as the first-selected agent of arteriosteogenesis treatment than women's arteria coronaria calcification degree of not taking hormone.The clinical research result shows that vitamin K alleviates angiosteosis and can also improve part patient's osteoporosis simultaneously, and the better tolerance of vitamin K treatment does not cause hypercoagulability.In that at present all can stop in the medicine of arteriosteogenesis progress, 3-hydroxyl-3-methylglutaric acid list acyl coenzyme A (3-hydroxyl-3-methyl glutaryl CoA, HMG CoA) reductase inhibitor may be the medicine that has potential most.HMG CoA reductase inhibitor may have special benefit to angiosteosis and osteoporosis and the patient who deposits, because HMG CoA reductase inhibitor can also suppress the formation of osteoclast and the absorption again of bone, and promotes the generation of new bone.
The application of ferric citrate in the prior art: at present ferric citrate is mainly the food additive of iron supplement or is nutritional supplement (irony reinforcement), for example, can make an addition to the milk powder of cookies, upgrading and wheat flour etc., and India also is used for Sal; Also be useful on the report of treatment chronic kidney hypofunction patient hyperphosphatemia, report ferric citrates such as Wu-Chang Yang can be used for the hemodialysis patients of chronic renal failure as new phosphorus bonding agent, itself and food are taken simultaneously, phosphate in can combining foods, reduce gastrointestinal tract to phosphatic absorption, thereby reduce chronic renal failure patient's hyperphosphatemia.Because also there is the phenomenon of cardiovascular calcifications in the chronic renal disease patient, there is viewpoint to think that hyperphosphatemia is the influence factor who causes ESRD (end-stage renal failure) patient cardiovascular calcifications, hyperphosphatemia has participated in the angioplany calcification of ESRD, but ESRD patient's serium inorganic phosphorus and the blood calcium that experimental results show that the part angiosteosis is still normal, and the angiosteosis and the human body serium inorganic phosphorus level that occur together such as atherosclerosis do not have direct relation in addition.Therefore, the cardiovascular system calcification of chronic kidney hypofunction is multi-pathogenesis, multipath, by the very complicated process that multiple molecule participates in, and the serium inorganic phosphorus level is to the unknown that influences of calcification.
Summary of the invention
The inventor finds after deliberation, ferric citrate can effectively be prevented and treated the arteriosteogenesis of the rat of being caused by vitamin D3 and nicotine, thereby proved that ferric citrate by intervening soft tissue calcification's forming process, can effectively suppress the cardiovascular heterotopic calcification, and might reverse the generation of heterotopic calcification.
The object of the present invention is to provide the new purposes of ferric citrate, that is, ferric citrate is in the application of the medicine of preparation treatment of vascular calcification.
The present invention also aims to provide a kind of new purposes of ferric citrate, especially, the application of ferric citrate in the medicine of preparation control cardiovascular system calcification relevant disease.
The invention provides the application of ferric citrate in the medicine of preparation treatment of vascular calcification.
Above-mentioned angiosteosis especially refers to the cardiovascular heterotopic calcification.
The invention provides a kind of new purposes of ferric citrate, promptly to the preventive and therapeutic effect of cardiovascular heterotopic calcification.
The present invention also provides the application of ferric citrate in the medicine of preparation control cardiovascular heterotopic calcification.
Wherein said cardiovascular heterotopic calcification comprises inner membrance calcification and/or middle film calcification, and described inner membrance calcification comprises the angiosteosis that betides under atherosclerosis or the related pathologies state; The film calcification comprises the angiosteosis under the pathological states such as betiding aging and diabetes, uremia in described.
Control cardiovascular heterotopic calcification of the present invention had both comprised prevention cardiovascular heterotopic calcification, also comprised treatment cardiovascular heterotopic calcification simultaneously, and wherein, prevention angiosteosis of the present invention comprises the complication that prevention is caused by angiosteosis, as hypertension etc.Experiment showed, that ferric citrate all can get a desired effect in the process of prevention and treatment heterotopic calcification.
Term in the context of the invention " prevention " not only refers to prevent fully a certain influence, also refers to before seizure of disease or the early stage any part of showing effect is prevented basically, weakens, reduces, gone down or eliminates this influence.
Term in the context of the invention " treatment " refers to any effect useful to the progress of disease, is included in the development that weakens, reduces, goes down after the seizure of disease or eliminate pathology.
The application of ferric citrate provided by the invention in the medicine of preparation treatment of vascular calcification, this application comprise ferric citrate the preparation control occur together by diseases such as aging, diabetes, end stagerenaldisease or atherosclerosiss or the medicine of the angiosteosis that causes in application.
The report that ferric citrate treatment chronic kidney hypofunction patient hyperphosphatemia was once arranged, and hyperphosphatemia is the influence factor who causes ESRD (end-stage renal failure) patient cardiovascular calcifications, hyperphosphatemia has participated in the angioplany calcification of ESRD, influence cardiovascular calcifications so there is viewpoint to think that ferric citrate is also permitted by reducing the serium inorganic phosphorus value, but of the present invention experimental results demonstrate, there be ESRD patient's the serium inorganic phosphorus and the blood calcium of part angiosteosis still normal, the mechanism that heterotopic calcification is described is complicated, though do not get rid of the influence that may have hyperphospheremia, but what can not query is, local factor (as PDGF) or inhibition calcification factor are (as fetuin-A, MGP etc.) also in action, for example, MGP substrate Gla albumen, deposition at local organization strong inhibition calcium microcosmic salt, the PDGF platelet derived growth factor can increase smooth muscle cell the picked-up of phosphorus not increased affinity to phosphorus, PDGF is also in the activation and the expression of inducing NPC with a kind of time and dose-dependent mode simultaneously, and the calcification of promotion smooth muscle cell, illustrate that the calcification of PDGF mediation can occur under the normal situation of serium inorganic phosphorus.The angiosteosis and the human body serium inorganic phosphorus level that occur together such as atherosclerosis do not have direct relation in addition.Therefore, the cardiovascular system calcification of chronic kidney hypofunction is multi-pathogenesis, multipath, by the very complicated process that multiple molecule participates in, and the serium inorganic phosphorus level is to the unknown that influences of calcification.
Employing of the present invention and the good rat VDN of cardiovascular system of human body calcification dependency animal model.Vitamin D and nicotine (VDN) cause the elastic artery calcification of adult rat, be and change maximally related angiosteosis animal model by the age vascular pathological relevant with the age, calbindin---the S-100 that in human artery's atherosclerotic lesion process, finds, relevant with film calcification in the VDN rat artery, the mechanism of VDN rat model arteriosteogenesis and result and the atherosis angiosteosis of human artery are quite similar, the basic feature of human artery's calcification is old-age group or relevant vascular lesion of age, as atherosclerosis, diabetes and kidney disease, and the most frequently used animal model of angiosteosis takes place in the rat pathological change that to be research relevant with old-age group and/or age etc.Adopt vitamin D and nicotine (VDN) to cause the intensity and the people of elastic artery calcification of adult rat close, can be used as and be used to study the calcification of people's blood vessel and cause arteriosclerotic animal model, specifically referring to Nathalie Niederhoffer, Yuri V.Bobryshev, et al.Aortic calcification produced by Vitamin D3 plus nicotine.Journal of vascular research 1997; 34:386-398.
For estimating ferric citrate at the effectiveness aspect the control cardiovascular system heterotopic calcification, the present invention does with following pharmacological effect experiment.
One, the ferric citrate administration is for the therapeutical effect of heterotopic calcification
1. material: male Sprague-Dawley (SD) rat, 150～180g provides the (quality certification number: SCXK capital 2002-0001) by Department Of Medicine, Peking University's Experimental Animal Center; Nicotine, vitamin D
3Available from Sigma company;
45CaCl
2Available from U.S. New England Nuclear company; (alkaline phosphatase ALP) measures test kit available from Beijing Li De Man to alkali phosphatase; All the other reagent are homemade analytical pure; Ferric citrate is provided by consonance pharmaceutcal corporation, Ltd, makes suspension with 4% starch solution, by different dosing dosage gastric infusion.
2. experimental technique
2.1 preparation of rat aorta calcification model and experiment grouping
With reference to the Niederhoffer method, preparation rat aorta calcification animal model.54 of male SD rats, body weight 150～180g is divided into 5 groups at random: (1) angiosteosis group (VDN) (n=12): test first day 9:00 and give rat intramuscular injection vitamin D
3(3 * 10
5U/kg) and nicotine be dissolved in soybean oil (25mg/kg 5ml/kg) irritate stomach, and the 18:00 nicotine repeats to irritate stomach 1 time, after this is left intact, and routine feeding is after 4 weeks, and every day 4%, starch solution was irritated stomach, continuous 4 weeks; (2) the blank group (control, n=10): give the intramuscular injection of equivalent soybean oil, simple soybean oil is irritated stomach and replaced vitamin D
3And nicotine, after this being left intact, routine feeding is after 4 weeks, and every day 4%, starch solution was irritated stomach, continuous 4 weeks; (3) angiosteosis+ferric citrate low dose group (VDN+L) is (n=12): after this calcification processing is left intact with (1), and routine feeding is after 4 weeks, and every day, according to dosage 370mg/kg gave ferric citrate, continuously 4 weeks of gastric infusion; (4) dosage group (VDN+A) is (n=10) in angiosteosis+ferric citrate: medication is with (3), and dosage is 740mg/kg; (5) angiosteosis+ferric citrate high dose group (VDN+H) is (n=10): medication is with (3), and dosage is the 1110mg/kg ferric citrate, continuously 4 weeks of gastric infusion.
Each treated animal is conventional raises, and surveys a body weight, and surveys tail arterial blood pressure weekly one time in 3 days; After 8 weeks, urethane 1g/kg intraperitoneal injection of anesthesia animal, blood is got on the optical fundus, measures blood parameters; Win heart and total length thoracoabdominal aorta, behind normal saline flushing, heart is weighed, and-70 ℃ store for future use.
2.2 von Kossa dyeing
Arch from aorta, get the thoracic aorta vascular strip of 1～1.5cm, fix with 4% paraformaldehyde; With paraffin embedding thoracic aorta and section, 7 μ m, conventional dewaxing, dehydration; Immerse 1% silver nitrate solution, behind the irradiation 30min, slide is put in 1min in 5% hypo solution under daylight, basic fuchsin returns and dyes; Dehydration, transparent, mounting, om observation.
2.3 tissue
45The Ca deposition
Get thoracic aorta (about 20mg), be prepared into the tissue slice about 0.3mm, place 1ml DMEM Incubating Solution, add 37kBq/mL
45CaCl
2, entire body integration number is 95%O
2-5%CO
2Gas is after lasting balance is hatched 10h in 37 ℃ of waters bath with thermostatic control, with distilled water flushing 3 times; Dry (80 ℃, 2h), weigh, get 0.5mL formic acid digestion tissue; Get the 0.4mL Digestive system and place scintillation vial, add the 4mL scintillation solution, survey with β-liquid scintillation counter
45Ca
2+Radioactivity (CPM/mg).
2.4 blood vessel calcium content
Get ventral aorta (about 10mg), dry (2-3h) for 80 ℃, weigh, be transferred in the quartz curette, add 2mol/L nitric acid 1ml, 2 perchloric acid, 180 ℃ of digestion are also dried, and redissolve with deionized water the cooling back, is settled to 2ml.Atomic emission spectrum reads absorbance at 422.7nm, is converted into the calcium content (μ mol/g dw) of tissue.
2.5 ALP (alkali phosphatase) determination of activity
Get the about 10mg of ventral aorta, ooze PBS with grade and prepare tissue homogenate (homogenate buffer: 20mmol/LHEPES, pH7.4,0.2%NP-40,20mmol/L MgCl
2, PBS is settled to 50ml).The centrifugal 10min of 8000 * g after the homogenate draws supernatant.The BCA method is carried out protein quantification.Press ALP and measure the test kit explanation, adopt the SFBC performance rate method to measure the ALP of vascular tissue activity [U/ (mgPro)].Concrete grammar: with testing sample, reagent 1, reagent 2 is pressed 1: 50: 10 mixed, and mixture, reads 2 minutes internal absorbances with microplate reader and changes after 60 seconds 37 ℃ of insulations at the 405nm place.ALP is active to be calculated as standard value with ρ-2 nitrophenol, generates the 1nmol nitrophenol in the 1 unit representation 30min.The result carries out standardization with protein content.
2.6 serium inorganic phosphorus, blood calcium are measured
Behind the rat anesthesia, blood is got on the optical fundus, adopts phosphomolybdic acid method and o-cresolphthalein complex copper (OCPC) method to measure serium inorganic phosphorus, blood calcium (mmol/L).
2.7 the rat tail arterial blood pressure is measured
Adopt RBP-1B type rat non-invasive blood pressure measuring to measure rat tail arterial blood pressure (mmHg), weekly.
2.8 statistical procedures: data are represented with means standard deviation, relatively adopt the t check between group, and there is statistical significance P＜0.05 for difference.
Rat heart muscle and aortic tissue general features (x ± s) is respectively organized in table 1 experiment
*Expression has been compared significant difference P＜0.05 with the VDN group; ## represents to have compared significant difference P＜0.05 with the control group
Rat arteria caudalis blood pressure (x ± s) is respectively organized in table 2 experiment
*Compared significant difference P＜0.05 with the VDN group; ## represents to have compared with matched group significant difference P＜0.05.
2.9 result:
(1) general features of rat aorta calcification
Compare with matched group, see Table 1, the blood vessel alkaline phosphatase activity high 1.9 times (P＜0.05) of the inductive VDN group of vitamin D3 and nicotine rat, vascular tissue's calcium content high 1.2 times (P＜0.05),
45Ca deposits high 1.7 times (P＜0.05), and the content of phosphorus has reduced by 10.68% (P＜0.05), calcium content there was no significant difference in the blood, H/BW ratio there was no significant difference in the blood plasma.
(2) ferric citrate is to the influence of rat calcification
Compare with model group (VDN group), see Table 2, the VDN+L group, the VDN+A group, the alkaline phosphatase activity of VDN+H group rat reduces by 3.96%, 7.45%, 25.52% respectively; Vascular tissue's calcium content reduces by 17.90% (P＜0.05), 4.65%, 2.93% respectively; The calcium deposition reduces by 19.26% (P＜0.05), 17.08%, 12.90% respectively; Each group of calcium phosphorus content is compared with the VDN group in the blood plasma does not then have significant difference, H/BW ratio there was no significant difference; Each tail arterial blood pressure of organizing rat reduces by 11.22%, 10.2%, 8.7% than the VDN group is average, all P＜0.05.
Two. ferric citrate is to the influence of rat blood pressure and serium inorganic phosphorus, blood calcium concentration
1. compare with the blank group, do not have significant change through blood pressure and blood calcium, the serium inorganic phosphorus concentration of vitamin D3 and the inductive rat VDN of nicotine model group; Compare with model group (VDN group), all around during the administration, except that VDN+ ferric citrate high dose group reduces 6.5% at second all blood pressures, P＜0.05, all the other each groups around in blood pressure do not have significant change, see Table 3.
The comparison of rat blood pressure measured value is respectively organized in table 3 experiment
*P＜0.05 is compared with normal group;
*P＜0.05 is compared with model group.
First week was respectively organized no significant difference between the blood pressure; Medicine is blank organizes second and third week respectively than normal group hypertension 8.5% and 11.5%, and significant difference is arranged; Heavy dose of second week of group reduces by 6.5% than model group blood pressure, and significant difference is arranged; Respectively organize there was no significant difference between the blood pressure around the.
2. compare with model group (VDN group), the blood calcium concentration of VDN+ ferric citrate low dosage, middle dosage, high dose group all raises, P＜0.05, and serium inorganic phosphorus concentration does not have significant difference, sees Table 4.
The comparison of rat serium inorganic phosphorus, blood calcium determination value is respectively organized in table 4 experiment
*P＜0.05 is compared with model group
The blood calcium aspect, each dosage group of medicine is higher by 52.4% respectively than model group blood calcium, 8%, 4.3%, model and blank there was no significant difference, the blank group of medicine is higher; Serium inorganic phosphorus content aspect, each dosage group of medicine is compared there was no significant difference with model group, does not also have tangible trend between each group, shows that ferric citrate is not is to reduce blood calcium by reducing serium inorganic phosphorus content as inferring in the industry.
Three. blood vessel calcium content and blood vessel determination of alkaline phosphatase activity (the prevention calcification of ferric citrate).
1. material: male Sprague-Dawley (SD) rat, 150～180g provides the (quality certification number: SCXK capital 2002-0001) by Department Of Medicine, Peking University's Experimental Animal Center; Nicotine, vitamin D
3Available from Sigma company;
45CaCl
2Available from U.S. New England Nuclear company; (alkalinephosphatase ALP) measures test kit available from Beijing Li De Man to alkali phosphatase; All the other reagent are homemade analytical pure.
2. method
2.1 preparation of rat aorta calcification model and experiment grouping
With reference to the Niederhoffer method, preparation rat aorta calcification animal model.50 of male SD rats, body weight 150～180g is divided into 5 groups at random: (1) angiosteosis group (VDN, n=10): test first day 9:00 and give rat intramuscular injection vitamin D
3(3 * 10
5U/kg) and nicotine be dissolved in soybean oil (25mg/kg 5ml/kg) irritate stomach, and the 18:00 nicotine repeats to irritate stomach 1 time, after this is left intact, and routine feeding is after 4 weeks, and every day 4%, starch solution was irritated stomach, continuous 4 weeks; (2) the blank group (control, n=10): give the intramuscular injection of equivalent soybean oil, simple soybean oil is irritated stomach and replaced vitamin D
3And nicotine, after this being left intact, routine feeding is after 4 weeks, and every day 4%, starch solution was irritated stomach, continuous 4 weeks; (3) angiosteosis+ferric citrate low dose group (VDN+L, or VDN+ is low) (n=10): calcification processing is with (1), and every day, according to dosage 370mg/kg gave ferric citrate, continuously 4 weeks of gastric infusion; (4) dosage group in angiosteosis+ferric citrate (VDN+A, or among the VDN+) (n=10): medication is with (3), and dosage is 740mg/kg; (5) (VDN+H, or VDN+ height, n=10): medication is with (3), and dosage is the 1110mg/kg ferric citrate, continuously 4 weeks of gastric infusion in angiosteosis+ferric citrate high dose group.
Each treated animal is conventional raises, and surveys a body weight, and surveys tail arterial blood pressure weekly one time in 3 days.After 4 weeks, urethane 1g/kg intraperitoneal injection of anesthesia animal, blood is got on the optical fundus, measures blood parameters; Win heart and total length thoracoabdominal aorta, behind normal saline flushing, heart is weighed, and-70 ℃ store for future use.
2.2von Kossa dyeing
Arch from aorta, get the thoracic aorta vascular strip of 1～1.5cm, fix with 4% paraformaldehyde.With paraffin embedding thoracic aorta and section, 7 μ m, conventional dewaxing, dehydration; Immerse 1% silver nitrate solution, behind the irradiation 30min, slide is put in 1min in 5% hypo solution under daylight, basic fuchsin returns and dyes; Dehydration, transparent, mounting, om observation.
2.3 blood vessel calcium content
Get the about 10mg of ventral aorta, dry (2-3h) for 80 ℃, weigh, be transferred in the quartz curette, add the nitric acid 1ml of 2mol/L, 2 perchloric acid, 180 ℃ of digestion are also dried, and redissolve with deionized water the cooling back, is settled to 2ml.Atomic emission spectrum reads absorbance at the 422.7nm place, is converted into the calcium content (μ mol/g dw) of tissue.
(2.4ALP alkaline phosphatase, alkali phosphatase) determination of activity
Get the about 10mg of ventral aorta, ooze PBS with grade and prepare tissue homogenate (homogenate buffer: 20mmol/LHEPES, pH7.4,0.2%NP-40,20mmol/L MgCl
2, PBS is settled to 50ml).The centrifugal 10min of 8000 * g after the homogenate draws supernatant.The BCA method is carried out protein quantification.Press ALP and measure the test kit explanation, adopt the SFBC performance rate method to measure the ALP of vascular tissue activity [U/ (mgPro)].Concrete grammar: with testing sample, reagent 1, reagent 2 is pressed 1: 50: 10 mixed, and mixture, reads 2 minutes internal absorbances with microplate reader and changes after 60 seconds 37 ℃ of insulations at the 405nm place.ALP is active to be calculated as standard value with ρ-2 nitrophenol, generates the 1nmol nitrophenol in the 1 unit representation 30min.The result carries out standardization with protein content.
2.5 serium inorganic phosphorus, blood calcium are measured
Behind the rat anesthesia, blood is got on the optical fundus, adopts phosphomolybdic acid method and o-cresolphthalein complex copper (OCPC) method to measure serium inorganic phosphorus, blood calcium (mmol/L).
2.6 the rat tail arterial blood pressure is measured
Adopt RBP-1B type rat non-invasive blood pressure measuring to measure rat tail arterial blood pressure (mmHg), weekly.
2.7 statistical procedures
Data are represented with means standard deviation, relatively adopt the t check between group, and P＜0.05 is for there being statistical significance.
Rat aorta calcium is respectively organized in table 5 experiment and blood vessel determination of alkaline phosphatase activity value compares
| Group | The blood vessel calcium content (μ g/g, dw) | Blood vessel alkaline phosphatase activities (U/mg pr) |
| Blank group (B) | 369.8±48.74 | 226.87±154.63 |
| Model group (M) | 616.9±100.5 | 632.45±229.89 ** |
| Small dose group (L) | 396.7±46.44 | 758.05±381.53 |
| Middle dosage group (A) | 491.9±39.30 | 560.36±281.39 |
| Heavy dose of group (H) | 365.7±51.90 | 696.91±698.23 |
Annotate: x ± s, n=10,
*P＜0.01, the calcification model group compares with blank group; ##P＜0.01,
Table 5 is the result show, with matched group relatively, raise respectively through blood vessel and the myocardial calcium content of vitamin D3 and the inductive angiosteosis rat of nicotine (VDN group).
The VDN treated animal of this experiment is compared with matched group: blood vessel calcium content and blood vessel ALP activity all significantly increase (P＜0.01).
Four, three months therapeutical effect of ferric citrate drug treatment for heterotopic calcification
1. material
Male Sprague-Dawley (SD) rat, 150～180g provides the (quality certification number: SCXK capital 2002-0001) by Department Of Medicine, Peking University's Experimental Animal Center.Nicotine, vitamin D
3Available from Sigma company;
45CaCl
2Available from U.S. New England Nuclear company; (alkaline phosphatase ALP) measures test kit available from Beijing Li De Man to alkali phosphatase; All the other reagent are homemade analytical pure.
2. method
2.1 preparation of rat aorta calcification model and experiment grouping
With reference to the Niederhoffer method, preparation angiosteosis animal model.54 of male SD rats, body weight 150～180g is divided into 5 groups at random: (1) angiosteosis model group (VDN) (n=12): test first day 9:00 and give rat intramuscular injection vitamin D
3(3 * 10
5U/kg) and nicotine be dissolved in soybean oil (25mg/kg 5ml/kg) irritate stomach, and the 18:00 nicotine repeats to irritate stomach 1 time, after this is left intact, and routine feeding is after 4 weeks, and every day 4%, starch solution was irritated stomach, continuous 12 weeks; (2) blank group (control) (n=10): give the intramuscular injection of equivalent soybean oil, simple soybean oil filling stomach replacement vitamin D3 and nicotine, after this be left intact, routine feeding is after 4 weeks, and every day 4%, starch solution was irritated stomach, continuous 12 weeks; (3) treatment group: [1] angiosteosis+ferric citrate low dose group (VDN+L) (n=12): the same model group of calcification processing, after this be left intact, routine feeding is after 4 weeks, every day, according to dosage 180mg/kg gave ferric citrate, continuously 12 weeks of gastric infusion; [2] dosage group (VDN+A) is (n=10) in angiosteosis+ferric citrate: medication is with (1), and dosage is 370mg/kg; [3] angiosteosis+ferric citrate high dose group (VDN+H) is (n=10): the same model group of medication, dosage are 740mg/kg.
Each treated animal is conventional raises, and surveys a body weight, and surveys tail arterial blood pressure weekly one time in 3 days.After 16 weeks, urethane 1g/kg intraperitoneal injection of anesthesia animal, blood is got on the optical fundus, measures blood parameters; Win heart and total length thoracoabdominal aorta, behind normal saline flushing, heart is weighed, and-70 ℃ store for future use.
2.2 tissue
45The Ca deposition
Get thoracic aorta (about 20mg), be prepared into the tissue slice about 0.3mm, place 1ml DMEM Incubating Solution, add 37kBq/mL
45CaCl
2, entire body integration number is 95%O
2-5%CO
2Gas is after lasting balance is hatched 10h in 37 ℃ of waters bath with thermostatic control, with distilled water flushing 3 times; Dry (80 ℃, 2h), weigh, get 0.5mL formic acid digestion tissue; Get the 0.4mL Digestive system and place scintillation vial, add the 4mL scintillation solution, survey with β-liquid scintillation counter
45Ca
2+Radioactivity (CPM/mg).
2.3 blood vessel calcium content
Get ventral aorta (about 10mg), dry (80 ℃ 2～3h), weigh, be transferred in the quartz curette, add the nitric acid 1ml of 2mol/L, 2 perchloric acid, 180 ℃ are nitrated and dry, and redissolve with deionized water the cooling back, is settled to 2ml.Atomic emission spectrum reads absorbance at the 422.7nm place, is converted into the calcium content (μ mol/gdw) of tissue.
2.4 ALP (alkali phosphatase) determination of activity
Get the about 10mg of ventral aorta, ooze PBS with grade and prepare tissue homogenate (homogenate buffer: 20mmol/LHEPES, pH7.4,0.2%NP-40,20mmol/L MgCl
2, PBS is settled to 50ml).The centrifugal 10min of 8000 * g after the homogenate draws supernatant.The BCA method is carried out protein quantification.Press ALP and measure the test kit explanation, adopt the SFBC performance rate method to measure the ALP of vascular tissue activity [U/ (mgPro)].Concrete grammar: with testing sample, reagent 1, reagent 2 is pressed 1: 50: 10 mixed, and mixture, reads 2 minutes internal absorbances with microplate reader and changes after 60 seconds 37 ℃ of insulations at the 405nm place.ALP is active to be calculated as standard value with ρ-2 nitrophenol, generates the 1nmol nitrophenol in the 1 unit representation 30min.The result carries out standardization with protein content.
2.5 serium inorganic phosphorus, blood calcium are measured
Behind the rat anesthesia, blood is got on the optical fundus, adopts phosphomolybdic acid method and o-cresolphthalein complex copper (OCPC) method to measure serium inorganic phosphorus, blood calcium (mmol/L).
2.6 the rat tail arterial blood pressure is measured
Adopt RBP-1B type rat non-invasive blood pressure measuring to measure rat tail arterial blood pressure (mmHg), weekly.
2.7 statistical procedures
Data are represented with means standard deviation, relatively adopt the t check between group.
2.8 the results are shown in Table 6 and 7.Treat trimestral experimental result and show, the calcification of ferric citrate treatment group obviously alleviates, and the blood pressure of model group is far above ferric citrate treatment group and normal group, and treatment group blood pressure has returned to normal level.
Table 6 calcification rat heart muscle and aortic tissue general features (x ± s)
*Expression has been compared significant difference P＜0.05 with the VDN group;
*Expression has been compared significant difference P＜0.01 with the VDN group;
* *Expression has been compared significant difference P＜0.005 with the VDN group.
Table 7. rat arteria caudalis blood pressure determination result (x ± s)
*Expression has been compared significant difference P＜0.05 with the VDN group;
*Expression has been compared significant difference P＜0.01 with the VDN group;
* *Expression has been compared significant difference P＜0.005 with the VDN group.
The result
1. ferric citrate is to rat aorta calcium content, the active influence of ALP
Ferric citrate can effectively reduce vitamin D3 and the inductive rat aorta calcification of nicotine medium vessels calcium content, suppresses alkali phosphatase (ALP) activity; Compare with the blank group, through the blood vessel calcium content rising 67% of vitamin D3 and the inductive angiosteosis rat of nicotine (VDN group), the blood vessel alkaline phosphatase activities is 2.79 times of blank group; Compare with model group, the blood vessel calcium content of ferric citrate low dose group, middle dosage group and high dose group reduces by 43.1% (P＜0.01) respectively, 27.3% (P＜0.01) and 45.9% (P＜0.01); Alkaline phosphatase activities reduces by 21.1% (P＜0.05) respectively, 16.0% and 55.2% (P＜0.01).
Employing of the present invention and the good rat VDN of cardiovascular system of human body calcification dependency animal model, specifically referring to Nathalie Niederhoffer, Yuri V.Bobryshev, et al.Aortic calcification produced byVitamin D3 plus nicotine.Journal of vascular research 1997; 34:386-398 adopts vitamin D and nicotine (VDN) to cause the elastic artery calcification of adult rat, and it is that the vascular pathological relevant with age and age changes relevant angiosteosis animal model.Calbindin--the S-100 that finds in human artery's atherosclerotic lesion process is relevant with film calcification in the VDN rat artery.The mechanism of VDN rat model arteriosteogenesis and result and the atherosis angiosteosis of human artery are quite similar.The basic feature of human artery's calcification is old-age group or relevant vascular lesion of age, as atherosclerosis, diabetes and kidney disease, in addition, rat is the most frequently used animal model that angiosteosis takes place for the research pathological change relevant with old-age group and/or age etc., adopt vitamin D and nicotine (VDN) to cause the intensity and the people of elastic artery calcification of adult rat close, can be used as and be used to study the calcification of people's blood vessel and cause arteriosclerotic animal model.
For estimating the effectiveness of ferric citrate control cardiovascular system heterotopic calcification, the present invention adopts vitamin D3 and the inductive rat VDN of nicotine model, give ferric citrate simultaneously setting up model respectively, investigate the influence of ferric citrate the angiosteosis of VDN rat model; After setting up model, give ferric citrate, investigate the therapeutical effect of ferric citrate angiosteosis.Result of study is as follows:
Ferric citrate can effectively reduce VDN rat model angiosteosis speckle generation, reduce the blood vessel calcium content, the process of angiosteosis is produced useful curative effect.For the VDN rat that produces angiosteosis, ferric citrate can reduce blood vessel and organize calcium content, effectively reduces calcification, keeps blood pressure simultaneously at normal level simultaneously.
Ferric citrate can effectively prevent and treat the cardiovascular system calcification, reduces the complication such as hypertension that angiosteosis causes simultaneously.
Description of drawings
Fig. 1: blank group von Kossa coloration result;
Fig. 2: model group von Kossa coloration result;
Fig. 3: ferric citrate low dose group von Kossa coloration result;
Fig. 4: dosage group von Kossa coloration result in the ferric citrate;
Fig. 5: ferric citrate high dose group von Kossa coloration result;
Fig. 6: the inductive angiosteosis model group of ferric citrate treatment rat VDN von Kossa coloration result;
Fig. 7: the small dose group von Kossa coloration result of the inductive angiosteosis of ferric citrate treatment rat VDN;
Fig. 8: the middle dosage group von Kossa coloration result of the inductive angiosteosis of ferric citrate treatment rat VDN;
Fig. 9: the heavy dose group von Kossa coloration result of the inductive angiosteosis of ferric citrate treatment rat VDN.
The specific embodiment
Embodiment 1: the preparation of ferric citrate
With reference to " preparation method that food additive handbook Chemical Industry Press records adopts hydrated ferric oxide. and citric acid reactions to prepare ferric citrate.
Take by weighing hydrated ferric oxide. 106.9g and 220.7g citric acid monohydrate through micronization processes, the flask that places band to stir, add 1000ml water, be heated to 60 ℃ with water-bath, continue stirring reaction 72 hours, reaction end is judged, draw the 1ml supernatant, add 10ml water, detect with spectrophotography, solution is clarified substantially and is promptly reached reaction end.
Is 1.26 with reaction solution being concentrated into the solution relative density below 60 ℃, gets brown thickness concentrated solution.
This concentrated solution gets ferric citrate crude product 307g at drying under reduced pressure below 60 ℃.
Above-mentioned ferric citrate is placed beaker, added the 300ml soak with ethanol 15 minutes, leach ethanol after, continue to add 300ml ethanol, repeated to soak 15 minutes, filter and promptly get the ferric citrate finished product at drying under reduced pressure below 60 ℃.
Embodiment 2: the ferric citrate acute toxicity test in mice
On the trial test basis, get 70 of Kunming mouses (19～21g, male and female half and half), fasting is divided into 7 groups after 15 hours at random, 10 every group.With 4% gelatinized corn starch the ferric citrate of embodiment 1 preparation is mixed with the even suspension of 7 kinds of concentration, 7 dosage groups are set, dosage is followed successively by from high to low: 10.00,8.00,6.40,5.12,4.10,3.28 and 2.62g/kg.Give mouse stomach respectively by 20ml/kg, observe and write down activity, feed, skin, mucosa, feces and the death condition of animal after the administration at once, and after administration, observe once every day at least in two weeks.Dead animal is carried out obduction, observe the situation of change of each histoorgan.Calculate median lethal dose(LD 50) with the Bliss method.The result sees table 8;
Each dosage treated animal death toll of table 8.
| Dosage (g/kg) | 10.00 | 8.00 | 6.40 | 5.12 | 4.10 | 3.28 | 2.62 |
| Female | 5 | 5 | 5 | 3 | 2 | 1 | 0 |
| Male | 5 | 5 | 3 | 4 | 3 | 2 | 0 |
| Amount to | 10 | 10 | 8 | 7 | 5 | 3 | 0 |
Calculate median lethal dose(LD 50) with the Bliss method, wherein:
LD
50＝4.2821g/kg；
The 95% credible 3.7019-4.8935g/kg of being limited to (seeing Table 9).
The oral ferric citrate acute toxicity of table 9. mice LD
50Computer chart (Bliss method)
| Dosage | Log10 dose | Number of animals | Death toll | Death toll | The experiment probability | Return probability |
| (g/kg) | [Log(D)] | (only) | (only) | (％) | Unit (Y) | Unit (Y) |
| 10.00 | 1.00000 | 10 | 10 | 100 | -- | 7.6011 |
| 8.00 | 0.90309 | 10 | 10 | 100 | -- | 6.9168 |
| 6.40 | 0.80618 | 10 | 8 | 80 | 5.8415 | 6.2324 |
| 5.12 | 0.70927 | 10 | 7 | 70 | 5.5240 | 5.5481 |
| 4.10 | 0.61278 | 10 | 5 | 50 | 5.0000 | 4.8667 |
| Dosage | Log10 dose | Number of animals | Death toll | Death toll | The experiment probability | Return probability |
| 3.28 | 0.51587 | 10 | 3 | 30 | 4.4760 | 4.1823 |
| 2.62 | 0.41830 | 10 | 0 | 0 | -- | 3.4933 |
Regression equation Y (Probit)=0.53937+7.0617Log (D)
Median lethal dose(LD 50) LD
50=4.2821g/kg
LD
50(Feiller correction) 95% fiducial limit=3.7019-4.8935g/kg
LD
5＝2.5046g/kg
LD
95＝7.3214g/kg
The LD of the oral ferric citrate of mice
50Be 4.28g/kg, animal does not have significant change at once after administration, and the appearance activity reduces after about 1 hour, and state is dispirited, reactions such as dyspnea.Begin to occur dead after about 1.5 hours, death all occurred in after the medication within 24 hours.Dead animal is through dissecting, and each internal organs shows no obvious abnormalities.
Embodiment 3: the research of ferric citrate treatment arteriosteogenesis
The ferric citrate of embodiment 1 preparation is made suspension with 4% starch solution, by different dosing dosage gastric infusion.
Experimental technique prepares rat aorta calcification animal model with reference to the Niederhoffer method.
54 of male SD rats, body weight 150～180g is divided into 5 groups at random: (1) angiosteosis group (VDN) (n=12): test first day 9:00 and give rat intramuscular injection vitamin D
3(3 * 10
5U/kg) and nicotine be dissolved in soybean oil (25mg/kg 5ml/kg) irritate stomach, and the 18:00 nicotine repeats to irritate stomach 1 time, after this is left intact, and routine feeding is after 4 weeks, and every day 4%, starch solution was irritated stomach, continuous 4 weeks; (2) the blank group (control, n=10): give the intramuscular injection of equivalent soybean oil, simple soybean oil is irritated stomach and replaced vitamin D
3And nicotine, after this being left intact, routine feeding is after 4 weeks, and every day 4%, starch solution was irritated stomach, continuous 4 weeks; (3) angiosteosis+ferric citrate low dose group (VDN+L) is (n=12): after this calcification processing is left intact with (1), and routine feeding is after 4 weeks, and every day, according to dosage 370mg/kg gave ferric citrate, continuously 4 weeks of gastric infusion; (4) dosage group (VDN+A) is (n=10) in angiosteosis+ferric citrate: medication is with (3), and dosage is 740mg/kg; (5) angiosteosis+ferric citrate high dose group (VDN+H) is (n=10): medication is with (3), and dosage is the 1110mg/kg ferric citrate, continuously 4 weeks of gastric infusion.
Each treated animal is conventional raises, and surveys a body weight, and surveys tail arterial blood pressure weekly one time in 3 days; After 8 weeks, urethane 1g/kg intraperitoneal injection of anesthesia animal, blood is got on the optical fundus, measures blood parameters; Win heart and total length thoracoabdominal aorta, behind normal saline flushing, heart is weighed, and-70 ℃ store for future use.
Conventional method von Kossa dyeing, tissue
45The Ca deposition is surveyed with β-liquid scintillation counter
45Ca
2+Radioactivity (CPM/mg) is surveyed blood vessel calcium content and ALP (alkali phosphatase) activity, and the result carries out standardization with protein content; Adopt phosphomolybdic acid method and o-cresolphthalein complex copper method to measure serium inorganic phosphorus, blood calcium.
The result shows the influence of ferric citrate to the rat calcification: compare with model group (VDN group), VDN+ ferric citrate low dose group, dosage group in the VDN+ ferric citrate, the alkaline phosphatase activity of VDN+ ferric citrate high dose group rat reduces by 3.96% respectively, 7.45%, 25.52%; Vascular tissue's calcium content reduces by 17.90% (P＜0.05), 4.65%, 2.93% respectively; The calcium deposition reduces by 19.26% (P＜0.05), 17.08%, 12.90% respectively; Each group of calcium phosphorus content is compared with the VDN group in the blood plasma does not then have significant difference, H/BW ratio there was no significant difference; Each tail arterial blood pressure of organizing rat reduces by 11.22%, 10.2%, 8.7% than the VDN group is average, all P＜0.05.
Ferric citrate is to the influence of rat blood pressure with serium inorganic phosphorus, blood calcium concentration: compare with the blank group, do not have significant change through blood pressure and blood calcium, the serium inorganic phosphorus concentration of vitamin D3 and the inductive rat VDN of nicotine model group; Compare with model group (VDN group), all around during the administration, except that VDN+ ferric citrate high dose group reduces 6.5% at second all blood pressures, P＜0.05, all the other each groups around in blood pressure do not have significant change.
The blood calcium aspect, each dosage group of medicine is higher by 52.4% respectively than model group blood calcium, 8%, 4.3%, model and blank there was no significant difference, the blank group of medicine is higher; Serium inorganic phosphorus content aspect, each dosage group of medicine is compared there was no significant difference with model group, does not also have tangible trend between each group, shows that ferric citrate is not is to reduce blood calcium by reducing serium inorganic phosphorus content as inferring in the industry.
The prevention calcification of ferric citrate: the result shows that the VDN treated animal of this experiment is compared with matched group, and blood vessel calcium content and blood vessel ALP activity all significantly increase (P＜0.01).
Experimental result is seen Fig. 1 to Fig. 9, Fig. 1-the 5th wherein, blank group, matched group and each dosage group von Kossa coloration result of ferric citrate, Fig. 6 is the inductive angiosteosis model group of a ferric citrate treatment rat VDN von Kossa coloration result, Fig. 7-the 9th, each dosage group von Kossa coloration result of the inductive angiosteosis of ferric citrate treatment rat VDN.
More than described the preferred embodiment for the present invention, so it is not in order to limit the present invention.Those skilled in the art can not depart from the improvement and the variation of category of the present invention and spirit to embodiment disclosed herein.
Claims (7)
1. the application of ferric citrate in the medicine of preparation treatment angiosteosis.
2. application according to claim 1, wherein said angiosteosis are the cardiovascular heterotopic calcification.
3. application according to claim 2, wherein said cardiovascular heterotopic calcification are inner membrance and/or middle film calcification.
4. application according to claim 1, wherein said angiosteosis is the old and feeble angiosteosis that occurs together or cause.
5. application according to claim 1, wherein said angiosteosis are the angiosteosis that diabetes occur together or cause.
6. application according to claim 1, wherein said angiosteosis are the angiosteosis that end stagerenaldisease occurs together or causes.
7. application according to claim 1, wherein said angiosteosis are the angiosteosis that atherosclerosis occurs together or causes.
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