CN102389567A - New application of intermedin 1-53 in preparation of treatment medicament for lipid metabolism disorder - Google Patents
New application of intermedin 1-53 in preparation of treatment medicament for lipid metabolism disorder Download PDFInfo
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Abstract
The invention discloses a new application of an intermedin 1-53 in the preparation of a treatment medicament for lipid metabolism disorder. The intermedin 1-53 can be used for preparing products for preventing and/or treating animal fat metabolism disorder, the lipid metabolism disorder is at least one of the following five diseases: atherosclerosis caused by fatty liver, hypertriglyceridemia, high fatty acid lipoproteinaemia, hypercholesterolemia and hyperlipidemia. An active ingredient of the product for preventing and/or treating animal fat metabolism disorder is the intermedin 1-53. In the invention, by using physiological saline solution of the intermedin 1-53 for continuously treating a mouse with high fat diet for four weeks, lipid accumulation of a liver of the mouse can be obviously reduced, the hyperlipidemia can be significantly improved, and the increase of atherosclerotic plaque also can be obviously inhibited.
Description
Technical field
The present invention relates to the new purposes of the plain 1-53 of intermediary, particularly the new purposes of the plain 1-53 of intermediary in preparation disorders of lipid metabolism medicine.
Background technology
Westernization and the popular of moving life style of sitting less along with China resident dietary structure significantly increase with the disorderly relevant great metabolic disease of glycolipid metabolism such as the generation of type 2 diabetes mellitus, obesity, hyperlipemia and fatty liver more.According to up-to-date Epidemiological study, China present about more than 300,000,000 has the patient of various disorders of lipid metabolism, and the overall prevalence of diabetes has reached 10.7 ‰, and prevalence is up to 27.5 ‰ in urban population.Different with western countries, in the numerous disorders of lipid metabolism diseases of China, hypertriglyceridemia and non-alcoholic fatty liver disease are modal two types.China's hypertriglyceridemia prevalence surpasses 11.9%, and prevalence is up to 15% for developed regions non-alcoholic fatty liver disease (NonAlcoholic Fatty Liver Disease is called for short NAFLD), and in obese people, the NAFLD prevalence is up to more than 50%.According to China's one line city, 1,000,000 healthy population data of " (second) Chinese residents nutrition and investigation of health conditions in 2002 " issue, NAFLD and hypertriglyceridemia have been that male resident is ranked front two, women resident and ranked the 9th and the tenth great metabolic disease.Increasing fatty liver is mainly NAFLD at present, accounts for 90% of fatty liver.The prevalence of NAFLD increases year by year with advancing age, and is very common at elderly population NAFLD, and the sickness rate of child NAFLD also significantly increases in China in addition, explained that China's hyperlipemia and NAFLD morbidity has obvious popularity.A large amount of evidences show: hypertriglyceridemia and high fatty acid mass formed by blood stasis (abbreviating hyperlipemia as) are the major incentive of NAFLD; And show as a kind of liver of metabolism syndrome; NAFLD and insulin resistant and type 2 diabetes mellitus reciprocal causation; Promote the generation and the development of glycolipid metabolism disorder disease, hypertension, cardiovascular and cerebrovascular disease jointly, become the very serious public health problem of China.Therefore, press for very much at present the medicine that China's independent intellectual property right is arranged to type 2 diabetes mellitus and non-alcoholic fatty liver disease exploitation.
Intermediary is plain, and (Intermedin is the newcomer of calcitonin-gene-related peptide (CGRP) family (ADM, CGRP, calcitonin and Diabetes-associated peptide) that finds in succession of the U.S. in 2004 and Japanese scholar IMD), and their gene has high homology.People IMD gene mapping is in No. 22 chromosomes; The IMD precursor protein that coding is made up of 148 amino acid residues; N-terminal is a signal peptide; A plurality of protease cutting site are arranged on the IMD precursor molecule, can be cut in vivo and contain 47 amino acid whose Intermedin1-47 (IMD1-47) and contain two active parts of 40 amino acid whose Intermedin8-47 (IMD8-47).The the 93rd and 94 at the prepro-IMD molecule is a pair of smart amino acid residue, is the degradation site of endogenous peptidase, and degradable is that prepro-AMD95-147 is the plain 1-53 (IMD1-53) of intermediary in vivo.The polypeptide that the plain 1-53 of people intermediary is made up of 53 amino acid residues; Its aminoacid sequence is shown in the sequence in the sequence table 1; This artificial synthetic peptide sequence is corresponding to people intermediary plain the 95th to 147 amino acids residue (Roh J, etal., J Biol Chem.2004 Feb 20; 279 (8): 7264-74.).
Summary of the invention
The new purposes that the purpose of this invention is to provide the plain 1-53 of a kind of intermediary.This new purposes is that the plain 1-53 of intermediary prevents and/or treats the application in the disorders of lipid metabolism product in preparation.
Said disorders of lipid metabolism is at least a in following five kinds of diseases: the atherosclerosis that fatty liver, hypertriglyceridemia, high fatty acid mass formed by blood stasis, hypercholesterolemia and hyperlipemia cause.
The said tallow metabolism disorder product that prevents and/or treats has following at least a characteristic:
1) reduces the animal livers accumulation of lipid, specifically can be and reduce the animal livers accumulation of lipid that high fat diet causes;
2) rising of inhibition animal livers content of triglyceride; Specifically can be the rising that suppresses the animal livers content of triglyceride that high fat diet causes;
3) suppress the rising of animal livers cholesterol level, specifically can be the rising that suppresses the animal livers cholesterol level that high fat diet causes;
4) alleviate the animal chylemia, specifically can be and alleviate the animal chylemia that high fat diet causes;
5) rising of C-VLDL and low-density lipoprotein cholesterol content in the inhibition animal blood slurry promotes the rising of animal blood slurry middle-high density lipoprotein cholesterol content simultaneously; Specifically can be the rising that suppresses C-VLDL and low-density lipoprotein cholesterol content in the animal blood slurry that high fat diet causes, promote the rising of animal blood slurry middle-high density lipoprotein cholesterol content simultaneously.
The said disorders of lipid metabolism product activity composition that prevents and/or treats is the plain 1-53 of intermediary.
Saidly prevent and/or treat the normal saline solution that the disorders of lipid metabolism product is the plain 1-53 of intermediary.
The said intermediary concentration of plain 1-53 in said normal saline is 5.978 μ mol/L.
The aminoacid sequence of the plain 1-53 of said intermediary is the sequence shown in the sequence 1 in the sequence table.
Said normal saline is the sodium-chloride water solution of 0.9% (mass fraction).
Experiment showed, that through of the present invention the plain 1-53 of said intermediary can be used for preparation and prevents and/or treats the application in animal (like people, Mus) the disorders of lipid metabolism product.
The present invention uses the normal saline solution of the plain 1-53 of intermediary to around the high fat diet mice continued treatment, can obviously reduce the accumulation of lipid of mouse liver, and significantly improve hyperlipemia, also can obviously suppress the increase of atheromatous plaque.
Description of drawings
Fig. 1 is the high fat diet mouse liver outward appearance after the IMD1-53 treatment.Wherein, be followed successively by from left to right: full diet group (apoE-/-), high fat diet group (apoE-/-+HFD), high fat diet+treatment group (apoE-/-+HFD+IMD).
Fig. 2 is the high fat diet mouse liver tissue slice hematoxylin-eosin staining light microscopic photographic view after the IMD1-53 treatment.Wherein, be followed successively by from top to bottom: full diet group (apoE-/-), high fat diet group (apoE-/-+HFD), high fat diet+treatment group (apoE-/-+HFD+IMD), left column is 200 times of enlarged drawings, 1000 times of enlarged drawings are classified on the right side as.
Fig. 3 is the high fat diet mouse liver tissue slice oil red O stain light microscopic photographic view after the IMD1-53 treatment.Wherein, be followed successively by from top to bottom: full diet group (apoE-/-), high fat diet group (apoE-/-+HFD), high fat diet+treatment group (apoE-/-+HFD+IMD), left column is 200 times of enlarged drawings, 1000 times of enlarged drawings are classified on the right side as.
Fig. 4 handles the blood fat outward appearance of mice for each.Wherein, be followed successively by from left to right: matched group (C57BL/6J mice), full diet group (apoE-/-), full diet+treatment group (apoE-/-+IMD), the high fat diet group (apoE-/-+HFD), high fat diet+treatment group (apoE-/-+HFD+IMD).
Fig. 5 is that rapidly and efficiently liquid chromatograph (FPLC) method mensuration is respectively handled the plasma lipoprotein collection of illustrative plates of mice.Wherein, vLDL is a VLDL, and LDL is that low density lipoprotein, LDL, HDL are high density lipoprotein.
The atherosclerosis of aorta speckle that Fig. 6 respectively handles mice for oil red O stain shows distributes.Wherein, from left to right be followed successively by: matched group (C57BL/6J), full diet group (apoE-/-), full diet+treatment group (apoE-/-+IMD), the high fat diet group (apoE-/-+HFD), high fat diet+treatment group (apoE-/-+HFD+IMD).
Fig. 7 handles the oil red O stain result of mouse aorta root section for each.Wherein, from left to right each row is followed successively by: full diet group (apoE-/-), high fat diet group (apoE-/-+HFD), high fat diet+treatment group (apoE-/-+HFD+IMD), every row are respectively 3 different sample result of this group.
Fig. 8 handles the area of aorta atherosclerotic plaque quantitative result of mice through oil red O stain for each.Wherein, left figure abscissa represent successively from left to right C57BL/6J matched group, apoE-/-full diet group, apoE-/-high fat diet group, apoE-/-high fat diet+treatment group; Right figure abscissa represent successively from left to right apoE-/-full diet group, apoE-/-full diet+treatment group; * represent to have compared significant difference (remarkable) at α=0.05 level difference through one factor analysis of variance check and full diet group (apoE-/-), # represent through one factor analysis of variance check with the high fat diet group (apoE-/-+HFD) compared significant difference (remarkable) at α=0.05 level difference.
Fig. 9 is the fasting glucose curve.Wherein, it is that full diet is fed that Chow represents matched group, and HFD represents the high fat diet group, and HFD+IMD represents high fat diet+treatment group.* expression is compared with the matched group (Chow) of corresponding time point and to be had significant difference (P＜0.05), and # representes to compare with the high fat enteral feeding group (HFD) of corresponding time point and has significant difference (P＜0.05).
Figure 10 is a glucose tolerance curve.Wherein, it is that full diet is fed that Chow represents matched group, and HFD represents the high fat diet group, and HFD+IMD represents high fat diet+treatment group.* expression is compared with the matched group (Chow) of corresponding time point and to be had significant difference (P＜0.05), and # representes to compare with the high fat enteral feeding group (HFD) of corresponding time point and has significant difference (P＜0.05).
Figure 11 is the insulin tolerance curve.Wherein, it is that full diet is fed that Chow represents matched group, and HFD represents the high fat diet group, and HFD+IMD represents high fat diet+treatment group.* expression is compared with the matched group (Chow) of corresponding time point and to be had significant difference (P＜0.05), and # representes to compare with the high fat enteral feeding group (HFD) of corresponding time point and has significant difference (P＜0.05).
The specific embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Test material used among the following embodiment is following:
Male C 57 BL/6 J mouse is available from Beijing Vital River Experimental Animals Technology Co., Ltd.;
Male C 57 BL/6 J mouse (the apoE of apo E (apoE) gene knockout
-/-), available from Beijing Vital River Experimental Animals Technology Co., Ltd.;
The plain 1-53 (IMD1-53) of intermediary, its aminoacid sequence is seen the sequence 1 in the sequence table, available from health peptide biotechnology (Beijing) company limited, article No. is 010-60;
Normal saline: the sodium-chloride water solution of 0.9% (quality percentage composition);
Osmotic pumps:
model 2004; DURECT Corp.; Cupertino; CA, USA;
It is 5.978 μ mol/L that IMD1-53 solution: IMD1-53 is dissolved in the concentration that normal saline makes IMD1-53;
The feed ingredient of full diet: protein 20 .3%, carbohydrate 66.0%, fat 5.0%;
1000 gram normal diet casein containing proteins (200 gram), DL-methionine (3 gram), corn starch (150 gram), sucrose (500 gram), cellulose BW200 (50 gram), Semen Maydis oil (50 gram), mineral mixture S10001 (35 gram), mixed vitamin V10001 (10 gram), adipokinetic hormone (2 gram).
The feed ingredient of high fat diet: protein 20 .0%, carbohydrate 50.0%, fat 21.0%; 1001.54 gram high lipid food casein containing protein (195 gram), DL-methionine (3 gram), corn starch (50 gram), maltodextrin 10 (100 gram), sucrose (341 gram), cellulose (50 gram), butterfat (200 gram), Semen Maydis oil (10 gram), mineral mixture S10001 (35 gram), calcium carbonate (4 gram), mixed vitamin V10001 (10 gram), adipokinetic hormone (2 gram), cholesterol (1.5 gram), ethoxy quinoline (0.04 gram).
Regular iletin: quick-acting neutral insulin injections, Jiangsu Wanbang Biological Pharmaceutical Co., Ltd., H10890001, concentration of insulin is 40U/ml.
The plain 1-53 of embodiment 1, intermediary (IMD1-53) is to the improvement effect of mice fatty liver
1, test method
Get 55 20 week age size apoE
-/-Mice is divided into four groups at random: full diet group (18), full diet+treatment group (10), high fat diet group (15), high fat diet+treatment group (12); The wild type big or small male C 57 BL/6 J mouse in 20 age in week of raising with full diet is matched group (9).Only contain the osmotic pumps of normal saline in the subcutaneous implantation of mice of matched group, full diet group and high fat diet group, the speed that makes osmotic pumps pump normal saline is 0.25 μ l/h; In the osmotic pumps that IMD1-53 solution has been poured in the subcutaneous implantation of mice of full diet+treatment group and high fat diet+treatment group in advance, the speed that makes osmotic pumps pump IMD1-53 is 300ng/kg/h.Implant surgery is accomplished two days later; Mice to matched group, full diet group and full diet+treatment group begins the full diet nursing; Mice to high fat diet group and high fat diet+treatment group begins the high fat diet nursing; Compare with the full diet nursing, high fat diet is fed increases by 16% fat and 0.15% cholesterol.
After feeding for 4 weeks, fasting is after 12 hours, and 10% chloral hydrate anesthesia mice is cut the abdominal cavity open, microspur camera liver general appearance (Fig. 1).Cutting liver organization weighs; Calculate liver weight (table 1); And cut the part liver organization and carry out paraffin and frozen section; Carry out hematoxylin-eosin and oil red O stain and identify (Fig. 2 and Fig. 3), and, measure triglyceride and content of cholesterol (table 1) with methanol chloroform extracting liver organization lipid components.
Table 1.IMD1-53 is to the quantitative result of high fat diet mice fatty liver
Annotate: * refers to compare at α=0.05 level difference through one factor analysis of variance and full diet group (apoE-/-) remarkable, # refer to through one factor analysis of variance and high fat diet group (apoE-/-+HFD) compare at α=0.05 level difference remarkable.
2, result of the test
Fig. 1 shows; The high fat diet group (apoE-/-+HFD) the liver outward appearance of mice is obviously turned white; Show its accumulation of lipid obviously more than full diet group (apoE-/-) mice, and through the high fat diet mice of 4 all IMD1-53 treatments (apoE-/-+HFD+IMD) the liver lipid obviously be less than untreated high fat diet group (apoE-/-+HFD).
Fig. 2 shows; The high fat diet group (apoE-/-+HFD) in the liver paraffin section hematoxylin eosin stain showed cell of mice more cavity structure is arranged; Show that its lipid within endothelial cells accumulates obviously more than full diet group (apoE-/-) mice, and through the high fat diet mice of 4 all IMD1-53 treatments (apoE-/-+HFD+IMD) in the hepatocyte cavity structure obviously be less than untreated high fat diet group (apoE-/-+HFD).
Fig. 3 shows; The high fat diet group (apoE-/-+HFD) the liver frozen section oil red O stain of mice shows has the positive fat of a large amount of oil red O stains to drip formation in the born of the same parents; Explain that its lipid within endothelial cells accumulates obviously more than full diet group (apoE-/-) mice, and through the high fat diet mice of 4 all IMD1-53 treatments (apoE-/-+HFD+IMD) in the hepatocyte the positive fat of oil red O stain drip obviously be less than untreated high fat diet group (apoE-/-+HFD).
Table 1 result shows, in mouse liver weight, the liver organization cholesterol level full diet+treatment group (apoE-/-+IMD) significantly be lower than full diet group (apoE-/-) in the mice; In mouse liver weight, the liver organization triglyceride and cholesterol level the high fat diet group (apoE-/-+HFD) in the mice all apparently higher than full diet group (apoE-/-), and high fat diet+treatment group (apoE-/-+HFD+IMD) all significantly be lower than in the mice high fat diet group (apoE-/-+HFD).
In sum, high fat diet can significantly increase the mouse liver accumulation of lipid, and after giving the IMD1-53 treatment, then can significantly reduce the liver accumulation of lipid that high fat diet causes.
1, test method
Get 55 20 week age size apoE
-/-Mice is divided into four groups at random: full diet group (18), full diet+treatment group (10), high fat diet group (15), high fat diet+treatment group (12) are matched group (9) with the male C 57 BL/6 J mouse of the wild type of full diet size in 20 age in week.Only contain the osmotic pumps of normal saline in the subcutaneous implantation of mice of matched group, full diet group and high fat diet group, the speed that makes osmotic pumps pump normal saline is 0.25 μ l/h; In the osmotic pumps that IMD1-53 solution has been poured in the subcutaneous implantation of mice of full diet+treatment group and high fat diet+treatment group in advance, the speed that makes osmotic pumps pump IMD1-53 is 300ng/kg/h.Implant surgery is accomplished two days later; Mice to matched group, full diet group and full diet+treatment group begins the full diet nursing; Mice to high fat diet group and high fat diet+treatment group begins the high fat diet nursing; Compare with the full diet nursing, high fat diet is fed increases by 16% fat and 0.15% cholesterol.
Fed for 4 weeks, fasting was obtained blood sample through endocanthion after 12 hours, in about 1ml blood, added 10%EDTA-Na
2Anticoagulant, 3000g got blood plasma in centrifugal 10 minutes.Directly take the blood plasma outward appearance (Fig. 4) of being got after 4 weeks of nursing through the microspur photographing unit, according to people's such as Yamaguchi Y method (J Chromatogr B Biomed Sci Appl.1998Sep 25; 716 (1-2): 57-64.), adopt liquid chromatograph (FPLC) analysed for plasma lipoprotein (comprising VLDL (vLDL), low density lipoprotein, LDL (LDL), high density lipoprotein (HDL)) composition (Fig. 5) rapidly and efficiently.Feed during 4 weeks, respectively at the 7th, 14,21 day, fasting was obtained blood sample through endocanthion after 12 hours, in about 100 microliters of blood, added 10%EDTA-Na
2Anticoagulant; 3000g got blood plasma in centrifugal 10 minutes; Add the 28th day plasma sample of getting; All adopt total plasma cholesterol to measure test kit, plasma triglyceride mensuration test kit, blood plasma low density lipoprotein, LDL (LDL) mensuration test kit and plasma high density lipoprotein level (HDL) and measure test kit (Zhongsheng Beikong Biological Science & Technology Co., Ltd.; 000000180,000000220,000000210 and 000000190) respectively the T-CHOL in the blood plasma (TC), total triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), HDL-C (HDL-C) are carried out quantitative assay; And deduct LDL-C and HDL-C through TC, calculate C-VLDL (vLDL-C) (table 2).
Each component content (mg/dL) in the blood plasma of each processed group mice of table 2.
Annotate: * refers to compare at α=0.05 level difference through one factor analysis of variance and full diet group (apoE-/-) remarkable, # refer to through one factor analysis of variance and high fat diet group (apoE-/-+HFD) compare at α=0.05 level difference remarkable.
2, result of the test
Among Fig. 4; Matched group (C57BL/6J) blood plasma water white transparency; Full diet group (apoE-/-) blood plasma is muddy; Full diet+treatment group (apoE-/-+IMD) the muddy degree of blood plasma is lower than full diet group (apoE-/-), the high fat diet group (apoE-/-+HFD) blood plasma is creamy white, high fat diet+treatment group (apoE-/-+HFD+IMD) the muddy degree of blood plasma be starkly lower than high fat diet+treatment group (apoE-/-+HFD+IMD).Explain high fat diet can significantly increase the weight of apoE-/-chylemia of mice, and through the IMD1-53 treatment, can alleviate to a certain extent high fat diet apoE-/-chylemia of mice.
Among Fig. 5; Blood plasma VLDL (vLDL) peak of each processed group by height be followed successively by on earth (apoE-/-+HFD)＞(apoE-/-+HFD+IMD)=(apoE-/-)＞(apoE-/-+IMD)＞C57BL/6J; Low density lipoprotein, LDL (LDL) peak by height be followed successively by on earth (apoE-/-+HFD)＞(apoE-/-+HFD+IMD)=(apoE-/-)=(apoE-/-+IMD)＞C57BL/6J, high density lipoprotein (HDL) peak by height be followed successively by on earth (apoE-/-+HFD+IMD)=C57BL/6J＞(apoE-/-+HFD)=(apoE-/-+IMD)＞(apoE-/-).Explain high fat diet can significantly increase apoE-/-mouse blood in the content of VLDL (vLDL) and low density lipoprotein, LDL (LDL); And through after the IMD1-53 treatment; Can obviously reduce high fat diet apoE-/-mouse blood in the content of VLDL (vLDL) and low density lipoprotein, LDL (LDL), the content of high density lipoprotein (HDL) is obviously increased.
Table 2 result shows; The apoE-of high fat diet/-mice (apoE-/-+HFD) in the blood plasma content of total triglyceride (TG), T-CHOL (TC), C-VLDL (vLDL-C) and low-density lipoprotein cholesterol (LDL-C) contrast C57BL/6J and apoE-/-remarkable rising all arranged; Simultaneously the content of HDL-C (HDL-C) contrasts C57BL/6J and significantly reduces, and than apoE-/-significantly raise; And through the high fat diet apoE-of IMD1-53 treatment/-mice (apoE-/-+HFD+IMD) HDL-C (HDL-C) content in the blood plasma apparently higher than without the high fat diet apoE-of IMD1-53 treatment/-mice (apoE-/-+HFD), all other (TG, TC, vLDL-C and LDL-C) content all significantly be lower than apoE-/-+HFD.
In sum, give IMD1-53 treatment, can alleviate to a certain extent apoE-/-chylemia (Fig. 4) of mice, reduce the content (Fig. 5) of vLDL in the blood plasma; High fat diet can significantly increase the weight of apoE-/-chylemia of mice, the content of vLDL-C in the blood plasma, LDL-C, HDL-C, TG and TC is significantly raise; And after giving the IMD1-53 treatment; The apoE-that high fat diet causes/-chylemia of mice obviously alleviated; The rising of the vLDL-C of blood plasma, LDL-C, TG and TC content all has remarkable decline; Promptly give the rising that IMD1-53 treatment can obviously suppress vLDL, LDL, TG and the TC content of chylemia that high fat diet causes, blood plasma, give the IMD1-53 treatment simultaneously after, the content of the HDL-C of the blood plasma that high fat diet causes then further raise (table 2).The above results shows that the IMD1-53 treatment can significantly improve hyperlipemia.
Embodiment 3, IMD1-53 are to the improvement effect of mice atheromatous plaque
1, test method
After embodiment 2 mices are got blood and put to death,, common carotid artery and branch, aorta and iliac artery and branch thereof are dissociated out through PBS and 4% paraformaldehyde perfusion; And aorta and all branches thereof are cut open from lesser curvature side; Be fixed on the black cake wax, after 4% paraformaldehyde is fixing, carry out oil red O stain; Show the atheromatous plaque size of endarterium, get aorta and directly take (Fig. 6) through the microspur photographing unit.Get the aortic root section and carry out 40 times of light microscopic scannings (Fig. 7).
After the above-mentioned aorta of cutting open is taken with the microspur camera; Adopt the AP (lesion of Quantity One software to aortic tunica intima; Red part) area and aorta (aorta; Whole inner membrances) area carries out quantitatively, and the statistical computation plaque area accounts for the percentage ratio of aorta area, and the area of aorta atherosclerotic plaque order of severity is analyzed (Fig. 8).
2, result of the test
Among Fig. 6; The atheromatous plaque of matched group redfree; ApoE-/-artery surface that gives the full diet mice a small amount of red atheromatous plaque that distributes; ApoE-/-mice that gives full diet and IMD1-53 treatment only has a small amount of red atheromatous plaque in bow portion and crotch artery surface; ApoE-/-give the high fat diet mice at whole aorta and crotch artery surface thereof a large amount of red atheromatous plaque that all distributes, apoE-/-mice that gives high fat diet and IMD1-53 treatment only has a small amount of red atheromatous plaque in bow portion and crotch artery surface.
Among Fig. 7; ApoE-/-give full diet mice, apoE-/-give high fat diet mice, apoE-/-all there is the male dyeing of oil red O in various degree in the aortic valve place that gives three groups of high fat diet and IMD1-53 treatment mices, the dye levels size is followed successively by: apoE-/-+HFD+IMD=apoE-/-＜apoE-/-+HFD.
The result of Fig. 6-8 shows; After the IMD1-53 treatment; ApoE-/-mice atherosclerotic plaque area has reducing to a certain degree, and after high fat diet feeds, and apoE-/-the atherosclerotic plaque area of mice significantly increases; After giving the IMD1-53 treatment, the atherosclerotic plaque that high fat diet causes increases by significantly inhibition.
Get 30 16 week age size male C 57 BL/6 J mouse; Be divided into 3 groups at random, 10 every group, one group is matched group (the full diet feedstuff of feeding); One group of high fat diet group (the high fat diet feedstuff of feeding), one group is high fat diet+treatment group (the high fat diet feedstuff of feeding).Three groups of mices are earlier according to after diet fed for 4 weeks separately, only contain the osmotic pumps of normal saline in the subcutaneous implantation of mice of matched group, full diet group, and the speed that makes osmotic pumps pump normal saline is 0.25 μ l/h; In the osmotic pumps that IMD1-53 solution has been poured in the subcutaneous implantation of mice of high fat diet+treatment group in advance, the speed that makes osmotic pumps pump IMD1-53 is 300ng/kg/h.Implant surgery is accomplished two days later, matched group, full diet group mice is begun full diet feed, and the mice of high fat diet+treatment group is begun high fat diet feed, and feeds with full diet and compares, and high fat diet is fed increases by 16% fat and 0.15% cholesterol.
1, blood sugar detection: from feeding beginning; Measure fasting glucose weekly once; The mensuration mode is: 8 beginnings fasting in the morning on every Tuesdays; The tail vein is got blood after 6 hours; (result is as shown in Figure 9 for Luo Shi diagnostic products (Shanghai) Co., Ltd., the ECS000094) blood sugar level of mensuration mice with vigor type Luo Shi blood sugar test paper (Accu-
Active Test Strip).The result shows that after high fat diet fed for 4 weeks, mouse blood sugar obviously raise, and treats for 4 weeks since giving subcutaneous IMD1-53 the 5th week, continues medication for the 3rd week and all 4 o'clock, can obviously suppress the fasting glucose concentration rising that high fat diet causes.
2, insulin assay: when mice is raised to the 8th week, get blood with ELISA method (RAT/MOUSE INSULIN ELISA KIT-96-Well Plate, MILLIPORE; 6 Research Park Drive; St.Charles, Missouri63304 U.S.A., EZRMI-13K) plasma insulin content (table 3); The result shows that high fat diet group mice plasma insulin content raises; And IMD1-53 treatment back blood plasma insulin content significantly descends, i.e. IMD1-53 treatment can suppress the plasma insulin content rising that high fat diet causes, explains that the IMD1-53 treatment can improve the hyperinsulinemia that high fat diet causes.
Table 3. is respectively handled the plasma insulin level (mean+SD) of mice
Annotate: * refers to compare at α=0.05 level difference through one factor analysis of variance and matched group (Chow) remarkable, and # refers to compare at α=0.05 level difference remarkable through one factor analysis of variance with high fat diet group (HFD).
3, insulin resistant detects: when mice is raised to the 8th week, carry out carbohydrate tolerance (GTT), insulin tolerance (ITT) and positive sugared hyperinsulinism clamp test.
1) carbohydrate tolerance (GTT) test: the mice fasting is after 12 hours; Carry out 20% (mass fraction) glucose solution according to the 3g/kg body weight and irritate stomach; Respectively at irritating behind the stomach 0 minute, 15 minutes, 30 minutes, 60 minutes, 90 minutes; (the same step 1) of method, the result is shown in figure 10 to get blood examination survey blood sugar level through the tail vein.The result shows, after high fat diet fed for 8 weeks, compares with the full diet group; The glycemic peaks that glucose is irritated the high fat diet group mice behind the stomach significantly raises; And its blood glucose decrease speed also significantly slows down, and in high fat diet+treatment group, compares with the high fat diet group; Its glycemic peaks and blood glucose decrease speed all have clear improvement, and explain that IMD1-53 treats can obviously improve high fat diet 4 weeks to feed the mouse glucose tolerance cause unusual.
2) insulin tolerance (ITT): the mice fasting is after 4 hours; According to 2U/kg body weight lumbar injection insulin; Respectively at behind the injection of insulin 0 minute, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes; (the same step 1) of method, the result is shown in figure 11 to get blood examination survey blood sugar level through the tail vein.The result shows; After high fat diet fed for 8 weeks, compare with the full diet group, the blood glucose decrease speed of the high fat diet group mice behind the lumbar injection insulin slows down; And its blood sugar recovery speed is significantly accelerated; And in high fat diet+treatment group, the blood sugar level of identical time point all is lower than the high fat diet group, explains that IMD1-53 treats can obviously improve high fat diet 4 weeks to feed the plain tolerance of the mouse islets cause unusual.
3) positive sugared hyperinsulinism clamp test: the mice fasting was weighed after 12 hours; Under the narcotism, make the bilateral cervical venous cannulation respectively, fixing intubate, and keep the total venous blood flow of neck unobstructed.The left side connects the micro-injection pump that insulin solutions (0.9% normal saline configuration, concentration is 270mU/ml) is housed, and it is 6mU/kg/min that the adjusting flow rate pump makes injection of insulin speed; The right side connects 20% (mass fraction) glucose solution.Before the start injection insulin, measure base state mouse blood sugar value, behind the start injection insulin; Per 10 minutes tail veins are measured blood glucose, and (method with step 1) once when blood glucose is lower than 120mg/dL, increases the injection speed of 20% glucose gradually; And record; Be stabilized in 120 ± 10mg/dL until blood sugar level, and kept 60 minutes, record glucose injection speed at this moment.Glucose injection speed is fast more, shows that the insulin sensitivity of mice is high more, otherwise then has insulin resistant.The result is as shown in table 4; The result shows that after high fat diet fed for 8 weeks, high fat diet group glucose injection speed obviously descended; And in high fat diet+treatment group; Compare with the high fat diet group, its glucose injection speed obviously increases, and explains that IMD1-53 treats can obviously improve high fat diet 4 weeks and feed the plain opposing of the mouse islets cause.
Respectively handle the glucose injection speed (mean+SD) of mice in the positive sugared hyperinsulinism clamp test of table 4.
Annotate: * refers to compare at α=0.05 level difference through one factor analysis of variance and matched group (Chow) remarkable, and # refers to compare at α=0.05 level difference remarkable through one factor analysis of variance with high fat diet group (HFD).
Claims (9)
1. the plain 1-53 of intermediary prevents and/or treats the application in the tallow metabolism disorder product in preparation.
2. application according to claim 1 is characterized in that: said disorders of lipid metabolism is at least a in following five kinds of diseases: the atherosclerosis that fatty liver, hypertriglyceridemia, high fatty acid mass formed by blood stasis, hypercholesterolemia and hyperlipemia cause.
3. application according to claim 1 and 2 is characterized in that: the said tallow metabolism disorder product that prevents and/or treats has following at least a characteristic:
1) reduces the animal livers accumulation of lipid;
2) rising of inhibition animal livers content of triglyceride;
3) rising of inhibition animal livers cholesterol level;
4) alleviate the animal chylemia;
5) rising of C-VLDL and low-density lipoprotein cholesterol content in the inhibition animal blood slurry promotes the rising of animal blood slurry middle-high density lipoprotein cholesterol content simultaneously.
4. application according to claim 3 is characterized in that:
Said liver accumulation of lipid is the liver accumulation of lipid that high fat diet causes;
The rising of said liver tg content is the rising of the liver tg content that causes of high fat diet;
The rising of said liver cholesterol level is the rising of the liver cholesterol level that causes of high fat diet;
Said chylemia is the chylemia that high fat diet causes;
The rising of said C-VLDL, low-density lipoprotein cholesterol and HDL-C content is the rising of the high fat diet C-VLDL, low-density lipoprotein cholesterol and the HDL-C content that cause.
5. according to arbitrary described application among the claim 1-4, it is characterized in that: the said disorders of lipid metabolism product activity composition that prevents and/or treats is the plain 1-53 of intermediary.
6. according to arbitrary described application among the claim 1-5, it is characterized in that: saidly prevent and/or treat the normal saline solution that the disorders of lipid metabolism product is the plain 1-53 of intermediary.
7. according to arbitrary described application among the claim 1-6, it is characterized in that: the said intermediary concentration of plain 1-53 in said normal saline is 5.978 μ mol/L.
8. according to arbitrary described application among the claim 1-7, it is characterized in that: the aminoacid sequence of the plain 1-53 of said intermediary is the sequence shown in the sequence 1 in the sequence table.
9. according to arbitrary described application among the claim 1-8, it is characterized in that: said normal saline is the sodium-chloride water solution of 0.9% (mass fraction).
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009046852A1 (en) * | 2007-09-11 | 2009-04-16 | Mondobiotech Laboratories Ag | Therapeutic uses of intermedin 47 and 53 peptides |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009046852A1 (en) * | 2007-09-11 | 2009-04-16 | Mondobiotech Laboratories Ag | Therapeutic uses of intermedin 47 and 53 peptides |
Non-Patent Citations (2)
| Title |
|---|
| 周芸 等: "中介素的生物学效应研究进展", 《医学综述》, vol. 16, no. 1, 31 January 2010 (2010-01-31), pages 13 - 16 * |
| 范小芳 等: "高脂血症性脂肪性肝病大鼠肝脏Intermedin的表达", 《胃肠病学和肝病学杂志》, vol. 17, no. 6, 30 June 2008 (2008-06-30), pages 489 - 491 * |
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