WO2022033152A1 - Utilisation de cilp2 dans la préparation d'un médicament destiné à amoindrir le vieillissement cardiaque et l'hypertrophie myocardique - Google Patents
Utilisation de cilp2 dans la préparation d'un médicament destiné à amoindrir le vieillissement cardiaque et l'hypertrophie myocardique Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
Definitions
- the invention belongs to the technical field of biomedicine, and particularly relates to the application of CILP2 in the preparation of medicines for improving cardiac aging and cardiac hypertrophy.
- Heart failure can occur after the aging of the heart causes changes in its structure. Unlike “systolic heart failure,” most age-related heart failure occurs when systolic function is normal and is called “diastolic heart failure.” For example, aging-induced left ventricular fibrosis, stiffness, and wall thickening can cause myocardial diastolic dysfunction, leading to heart failure.
- the number of cardiomyocytes may decrease with age, but cardiac fibroblasts, which produce extracellular matrix and collagen, proliferate significantly.
- cardiac fibroblasts proliferate excessively, the synthesis and degradation of collagen will be unbalanced, the proportion of collagen will be unbalanced, the arrangement of cells will be disordered, and the deposition of extracellular matrix will occur.
- Collagen accumulation leads to interstitial fibrosis in the atria, stellate ganglia, and ventricles of the elderly, which in turn causes myocardial systolic and diastolic dysfunction.
- the CILP2 gene is located in band 11 (19p13.11) of the short arm 13 region of chromosome 19, with a length of 8395 bp.
- the protein encoded by the CILP2 gene is called cartilage intermediate layer protein 2 (cartilage intermediate layer protein 2).
- CILP2 is highly homologous to CILP1. As a massively secreted glycoprotein present in extracellular fluid, CILP1 is thought to play a role in the scaffolding of cartilage, and its dysfunction may lead to cartilage diseases. Studies have shown that CILP1 is expressed in the heart and muscle of adult mice, suggesting that it may function outside the cartilage.
- CILP2 also exists as a glycoprotein in human articular cartilage and ultrastructure, and may be involved in the formation of collagen VI in ultrastructure. Both CILP-1 and CILP-2 proteins can be cleaved into N-terminal fragments and C-terminal fragments at the furin endoprotease site. It is inferred from the protein structure and function: the N-terminal WxxW sequence is involved in the interaction of CILP with TGF- ⁇ play a key role ( Figure 1).
- the C-terminus has a homology of more than 90.9% with nucleotide pyrophosphohydrolase (NTPPHase), which may be related to the activity of nucleotide pyrophosphodiesterase (NPP), but it lacks the NPP catalytic site or bivalent cationic binding site.
- NPPPHase nucleotide pyrophosphohydrolase
- the present invention provides the application of CILP2 in the preparation of medicines for improving cardiac aging and cardiac hypertrophy, aiming to solve some of the problems in the prior art or at least alleviate some of the problems in the prior art.
- the invention provides an application of CILP2, which is mainly reflected in the application of CILP2 in the preparation of a medicine for preventing, relieving and/or treating cardiac hypertrophy caused by aging.
- the present invention is specifically the application of CILP2 in the preparation of a medicine for improving and/or treating cardiac aging and/or cardiac hypertrophy.
- CILP2 is a C-terminal fragment of CILP2 cleaved at the furin endoprotease site.
- the drug includes a secretagogue or agonist of CILP2.
- a drug for preventing, relieving and/or treating cardiac aging and/or cardiac hypertrophy comprising at least one of CILP2, a secretagogue or agonist of CILP2.
- the CILP2 secretagogue or agonist includes at least one of an overexpression plasmid of the CILP2 gene and an agonist capable of promoting the expression of CILP2.
- CILP2 secretagogue or agonist in the preparation of a medicament for improving and/or treating cardiac aging and/or cardiac hypertrophy.
- CILP2 gene and/or the expression product of CILP2 gene as a drug target in the preparation of a drug for improving and/or treating cardiac aging and/or cardiac hypertrophy.
- the present invention performs protein spectrum detection on the plasma of the aging population and the young population, and finds that the level of CILP2 in the blood circulation of the aging population decreases. Furthermore, in the results of protein spectrum detection in the plasma of the aging population and the young population, the peptide identification list shows that the 7 peptides marked in the mass spectrometry were compared with the full-length amino acid sequence of CILP2, and it was found that these peptides were all located in the C of the gene. end fragment.
- the invention establishes a mouse aging model by naturally rearing 22-month-old wild-type mice, using 8-week-old young control wild-type C57BL/6 mice, 22-month-old aging control mice and 22-month-old aging CILP2 overexpression mice as experiments object.
- the mouse cardiac ultrasound examination, cardiac weighing, myocardial fibrosis, cardiomyocyte size detection, cardiac hypertrophy markers and cardiac aging markers were performed.
- the results showed that compared with the young mice, the aged control mice had significantly thicker ventricular wall thickness, aggravated myocardial fibrosis, increased cross-sectional area of cardiomyocytes, and significantly increased cardiac hypertrophy indexes, including aging markers P16, P21, and P53. increased expression.
- the overexpression of CILP2 in the aging experimental group can reduce the thickness of the ventricular wall, reduce the weight of the heart, and the size of the cardiomyocytes is also significantly reduced.
- the expression levels of , P21 and P53 decreased, and the expression levels of inflammatory factors IL-1 ⁇ , IL-6, and TNF- ⁇ in the myocardium also decreased, and the trend changed to a younger age.
- the above results indicate that aging can promote the occurrence of aging cardiac hypertrophy, and overexpression of CILP2 can improve aging cardiac hypertrophy by improving the expression of aging marker genes, and provide new targets and strategies for the prevention, mitigation and/or treatment of aging-induced cardiac hypertrophy. Provided the theoretical basis and clinical basis.
- the research of the present invention proves that in an aging model, overexpression of CILP2 can inhibit the aging of the heart, reduce myocardial hypertrophy and fibrosis, and especially improve the effect of aging myocardial hypertrophy.
- CILP2 has the effect of improving cardiac aging and cardiac hypertrophy; based on the function of CILP2 in cardiac aging and cardiac hypertrophy, it provides a target for the development of drugs for the treatment of cardiac aging and cardiac hypertrophy;
- the agonists of CILP2 can be used for the preparation of medicaments for preventing, alleviating and/or treating cardiac aging and cardiac hypertrophy.
- Fig. 1 is the CILP2 gene structure diagram
- FIG. 2 is a mouse processing flow
- Figure 3 shows that the level of CILP2 transcription in myocardium is up-regulated after virus transfection. **: p ⁇ 0.01; ns: no statistical difference;
- Figure 4 shows the changes of cardiac structure and function in mice detected by cardiac ultrasound, as well as the changes of cardiac weight in mice;
- A typical M-mode diagram of cardiac ultrasound;
- B analysis results of LVPWs, LVPWd and EF values detected by cardiac ultrasound;
- C mouse Whole heart weight analysis, heart weight to body weight/tibia length ratio analysis, left and right ventricular weight analysis. ***: p ⁇ 0.001; **: p ⁇ 0.01; *: p ⁇ 0.05; ns: no statistical difference;
- Figure 5 shows HE staining and wheat germ agglutinin (WGA) staining of myocardial tissue sections.
- A HE staining of heart tissue sections, representative images under light microscope at 400 times;
- B WGA staining of heart tissue sections, representative images at 200 times and 400 times of light microscope;
- C RT-PCR detection of myocardial hypertrophy markers Myh6 and Myh7 changes at the mRNA level.
- Figure 6 shows the changes in the levels of markers P16, P21 and P53 of myocardial aging detected by RT-PCR and the level of cardiac inflammation in mice.
- A RT-PCR to detect the changes in the levels of markers P16, P21 and P53 of myocardial aging;
- B RT-PCR to detect the level of cardiac inflammation in mice.
- the normal temperature in the following embodiments of the present invention refers to the natural room temperature conditions in the four seasons without additional cooling or heating treatment. Generally, the normal temperature is controlled at 10-30°C, preferably 15-25°C.
- the invention discloses the application of CILP2 in the preparation of medicines for improving cardiac aging and cardiac hypertrophy.
- the species, gender, size and source of the experimental animals involved in the present invention C57BL/6, male mouse, 8-week-old young wild-type mouse, and 22-month-old aged wild-type mouse.
- the experimental mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.
- the present invention takes C57BL/6 as the experimental object, uses AAV9 virus to overexpress the CILP2 gene at the transcription level, and establishes a model of cardiac senescence and cardiac hypertrophy by rearing for 22 months.
- Expression of CILP2 can significantly reduce the levels of cardiac aging indicators P16, P21 and P53 in aging mice, reduce the levels of myocardial inflammatory factors IL-1 ⁇ , IL-6 and TNF- ⁇ , and the expression level of myocardial hypertrophy marker Myh7 in aging mice. And increase the expression level of Myh6, the thickness of the posterior wall of the heart is reduced, the degree of fibrosis is weakened, and the cardiomyocytes are reduced.
- CILP2 can regulate the occurrence of cardiac aging and cardiac hypertrophy, and has a protective effect on cardiac aging and cardiac hypertrophy. clinical basis. Therefore, in view of the above functions of CILP2, CILP2 can be used as a drug target to screen drugs for the treatment of cardiac aging and cardiac hypertrophy.
- the numbers of CILP2 (human) involved in the present invention in the NCBI database gene ID is 148113, nucleotide sequence number NM_153221.2, protein sequence NP_694953.2.
- mice used in the research were raised in the barrier environment of the Animal Experiment Center of the People's Hospital of Wuhan University, with constant temperature (20-26°C), constant humidity (40-70%), low noise ( ⁇ 60 decibels), and ventilation frequency of 15 times in the barrier. 12/12 hours light/dark cycle, 4 animals per cage were housed, and mice were fed ad libitum. Natural rearing to 22 months of age to build an aging model.
- the pHBAAV-CMV-MCS-3flag-T2A-ZsGreen plasmid vector was used to construct a true CILP2C fragment (sequence shown in SEQ ID NO.21).
- the nuclear expression plasmid was packaged as adeno-associated virus AAV9-CILP2, while the empty pHBAAV-CMV-MCS-3flag-T2A-ZsGreen plasmid was packaged as adeno-associated virus AAV9-Neg as a control.
- mice were injected with AAV9 virus via tail vein when they were 16 months old: adenovirus AAV9-Neg in control group and adenovirus AAV9-CILP2 in CILP2 overexpression group, the dose of virus per mouse injection was 5.5 ⁇ 10 11 particles, as shown in Figure 2 .
- RT-PCR Real-time PCR
- RNA extraction Add 1 ml of Trizol solution to the collected cardiomyocytes, pipet repeatedly on ice until the cardiomyocytes are fully lysed, let stand for 5 minutes, add 0.2 ml of chloroform, shake vigorously for 15-30 seconds, let stand for 2-3 minutes, Centrifuge at 12000rpm x 15min at 4°C. Pipette the aqueous layer into a new EP tube, add 0.5ml of isopropanol, mix the liquid in the tube gently, let stand for 10 minutes at room temperature, and centrifuge at 12000rpm x 10min at 4°C.
- RNA Discard the supernatant, add 1 ml of pre-cooled 75% ethanol to the pellet, resuspend the pellet and wash thoroughly, and centrifuge at 12000rpm x 5min at 4°C. The supernatant was discarded, air-dried, and an appropriate amount of DEPC water was added to solubilize the RNA at 65°C. Take 2ul RNA for agarose gel electrophoresis to check whether the RNA is extracted successfully, and store the remaining RNA at -80°C when not in use.
- RT-PCR Real-time fluorescence quantitative PCR
- RNA was extracted from the heart tissue of mice in each group, and cDNA was obtained after reverse transcription. The 2 method was used to specifically detect the mRNA expression level of CILP2 to verify whether CILP2 can be specifically up-regulated by AAV9-CILP2 virus.
- the results are shown in Figure 3.
- the transfection virus was identified at the transcriptional level.
- the CILP2 mRNA level in cardiomyocytes in the CILP2 overexpression group was about 10 times higher than that in the control group.
- the experiment was divided into three groups: 8-week-old young control group (Young control), aging control group (Old-AAV9-Neg), and aging CILP2 overexpression group (Old-AAV9-CILP2).
- Young control 8-week-old young control group
- Old-AAV9-Neg aging control group
- Old-AAV9-CILP2 aging CILP2 overexpression group
- HE staining method paraffin section I xylene for 2-5 minutes, II xylene for 2-5 minutes, 100% ethanol for 1 min, 95% ethanol for 1 min, 80% ethanol for 1 min, 70% ethanol for 1 min, first wash with tap water and then distilled water for 0.5 min, Harris' hematoxylin liquid 5min. Hematoxylin is a basic dye that can stain cell nuclei.
- 1% acidic alcohol a mixture of 1 mL of HCl and 99 mL of 70% alcohol
- the carbolic acid was washed with I xylene for 1 min and made it more transparent. After passing through II xylene for 1 min, the sections were taken out from the xylene, and the xylene other than the sections was wiped off with dry gauze. Results: The nucleus was blue-purple and the cytoplasm was red.
- Dewaxing the paraffin sections to water Place the sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and distilled water for washing.
- DAPI counterstained cell nuclei the slides were placed in PBS (PH7.4) and washed 3 times with shaking on a destaining shaker, 5 min each time. After the sections were slightly dried, DAPI staining solution was added dropwise to the circle, and incubated at room temperature for 10 min in the dark.
- Cover slides The slides were placed in PBS (PH7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After drying, the sections were mounted with anti-fluorescence quenching mounting medium.
- Wheat germ agglutinin can specifically bind to the myocardial cell membrane.
- the combination of the two can stain the myocardial cell membrane, and observe the stained cells in the myocardium through a microscope, so as to measure the diameter and area, and analyze whether the myocardial cells are hypertrophic.
- FIG. 5B cardiomyocytes in aging mice were significantly enlarged, and overexpression of CILP2 could significantly reduce the size of cardiomyocytes.
- the total RNA was extracted from the heart tissues of mice in each group, and the mRNA expression levels of Myh6 and Myh7, the indicators of cardiac hypertrophy, were detected by the method of 2.
- mice were sacrificed by cervical dislocation, the hearts were taken out and washed with PBS. After the whole body was weighed, the left and right ventricles were separated and weighed separately. The weight of the mouse heart was detected, as shown in Figure 4C, after the intervention of the expression level of CILP2, the weight of the left and right ventricles of the heart was reduced.
- Total RNA was extracted from the heart tissue of mice in each group, and the mRNA expression levels of aging-related factors P16, P21, and P53, and the mRNA expression levels of inflammatory factors IL-1 ⁇ , IL-6, and TNF- ⁇ were detected by two methods.
- P16, P21 and P53 are indicators of cardiac aging, and the expression levels of these three genes in the myocardium increase with age, and the expression levels of P16, P21 and P53 in the myocardium are reversed after up-regulation of CILP2 expression.
- chronic inflammation is a feature and pathogenic factor of aging
- the expression levels of inflammatory factors IL-1 ⁇ , IL-6 and TNF- ⁇ in the myocardium of three groups of mice were also detected in this application, and it was found that inflammatory factors in aging mice were The level of inflammatory factors in the myocardium was down-regulated after up-regulation of CILP2 expression.
- up-regulation of CILP2 expression may improve cardiac aging.
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