CN111840514B - Application of CILP2 in the preparation of drugs for improving cardiac aging and cardiac hypertrophy - Google Patents
Application of CILP2 in the preparation of drugs for improving cardiac aging and cardiac hypertrophy Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and particularly relates to application of CILP2 in preparation of a medicine for improving heart aging and myocardial hypertrophy. The invention takes a mouse as an experimental object, uses AAV9 virus to perform transcription level overexpression on CILP2 gene, and performs comparative research by establishing heart aging and myocardial hypertrophy models. The results show that compared with the aged wild type control group, the over-expression of CILP2 can obviously reduce the index level of heart aging of aged mice, reduce the index level of myocardial inflammation factors of aged mice, and improve the expression level of markers of myocardial hypertrophy, the wall thickness of the heart is reduced, the fibrosis degree is weakened, and myocardial cells are reduced. The results prove that the expression level of CILP2 can regulate the occurrence of heart aging and myocardial hypertrophy, has protective effects on the heart aging and the myocardial hypertrophy, and can provide theoretical basis and clinical basis for researching new targets and new strategies for preventing and treating the heart aging and the myocardial hypertrophy.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of CILP2 in preparation of a medicine for improving heart aging and myocardial hypertrophy.
Background
Health, aging and longevity are the most important concerns and the mysteries of people have been explored since ancient times. With the progress of China into aging society, diseases brought by aging seriously threaten the health of human beings, but the understanding of aging is still in the initial stage at present. Among the diseases caused by aging, cardiovascular diseases have been high leaders in the ranking of lethal causes over the years, in which heart failure is one of the most common causes of death. Recently, at the protein level, GDF11 and TIMP2 were identified as potential anti-aging factors. The application of anti-aging factors to aging body can slow down the aging progress of organs, and improve heart aging, cardiac hypertrophy and other organ functions of aging mice. Therefore, aging marker genes and proteins are actively searched for, which may predict the aging state of the body, and may become important targets for resisting aging and myocardial hypertrophy.
With age, the heart undergoes changes in its structure, function, cellular and molecular level, highlighted by myocardial hypertrophy, fibrosis, misfolded protein accumulation, mitochondrial dysfunction, decreased calcium channel activity and increased sympathetic nerve activity, which are also characteristic of heart aging, even in the absence of significant cardiovascular disease. Heart failure can occur after the heart aging causes its structural changes. Unlike "systolic heart failure", most age-related heart failure occurs under normal systolic function, called "diastolic heart failure". For example, aging-induced left ventricular fibrosis, stiffness and wall thickening all contribute to myocardial diastolic dysfunction, leading to heart failure. In an aging heart, the number of cardiomyocytes may decrease with age, but cardiac fibroblasts proliferate significantly, and produce extracellular matrix and collagen. When the cardiac fibroblasts proliferate excessively, imbalance of collagen synthesis and degradation, imbalance of collagen proportion, disorder of cell arrangement and extracellular matrix deposition occur, which are common pathological changes of many cardiovascular diseases at a certain stage. The accumulation of collagen leads to interstitial fibrosis of the atria, stellate ganglia and ventricles of the elderly, which in turn causes myocardial contraction and relaxation dysfunction.
At present, the pathogenesis of heart aging and myocardial hypertrophy caused by the heart aging is not completely clarified, and an effective medicament treatment means is lacking clinically. Therefore, exploring the molecular mechanism of the pathological occurrence of heart aging and aging myocardial hypertrophy is helpful to search for a new drug action target, improve the treatment scheme of resisting heart aging and aging myocardial hypertrophy and promote the development of accurate medical treatment in the field of heart aging prevention and treatment.
The CILP2 gene is located in the 11 band (19p13.11) of the short arm 13 region of chromosome 19, is 8395bp long, and the protein coded by the CILP2 gene is called cartilage intermediate layer protein 2(cartilage intermediate layer protein 2). CILP2 is highly homologous to CILP 1.CILP 1, which is present in extracellular fluid as a large number of secreted glycoproteins, is thought to function as a scaffold for cartilage, and its dysfunction may lead to cartilage disease. Studies have shown that CILP1 is expressed in the heart and muscle of adult mice, suggesting a possible extrachondral role. CILP2 is also present as a glycoprotein in human articular cartilage and ultrastructures, and may be involved in the formation of ultrastructural collagen VI. Both CILP-1 and CILP-2 proteins can be cleaved into N-terminal and C-terminal fragments at the furin cleavage protease site, which are presumed based on protein structure and function: the N-terminal WxxW sequence plays a key role in CILP interaction with TGF-. beta.s (FIG. 1). The homology of the C terminal and nucleotide pyrophosphate hydrolase (NTPPHase) is more than 90.9 percent, which is probably related to the activity of nucleotide pyrophosphate diesterase (NPP), but the C terminal lacks an NPP catalytic site or a divalent cation binding site.
Disclosure of Invention
In view of the problems of the prior art, the present invention provides the use of CILP2 in the manufacture of a medicament for improving heart aging and myocardial hypertrophy, and aims to solve at least some of the problems or at least alleviate some of the problems of the prior art.
The invention provides an application of CILP2, which is mainly embodied in the application of CILP2 in preparing a medicament for preventing, relieving and/or treating myocardial hypertrophy caused by aging.
The invention particularly relates to application of CILP2 in preparing a medicament for improving and/or treating heart aging and/or myocardial hypertrophy.
Further, the CILP2 is a C-terminal fragment of CILP2 cleaved at the furin cleavage protease site.
Further, the medicament comprises an secretagogue or agonist of CILP 2.
A medicament for preventing, ameliorating and/or treating heart aging and/or myocardial hypertrophy comprising at least one secretagogue or agonist of CILP2, CILP 2.
Further, the secretagogue or agonist of CILP2 includes at least one of an overexpression plasmid of CILP2 gene and an agonist capable of promoting CILP2 expression.
Use of an secretagogue or agonist of CILP2 for the preparation of a medicament for the amelioration and/or treatment of heart aging and/or myocardial hypertrophy.
The application of the CILP2 gene and/or CILP2 gene expression product as a drug target in preparing drugs for improving and/or treating heart aging and/or myocardial hypertrophy.
According to the invention, the plasma of the aged people and the plasma of the young people are subjected to protein mass spectrum detection, and the level of CILP2 in the blood circulation of the aged people is found to be reduced. And further, in the results of protein mass spectrometry detection of the blood plasma of the aged population and the young population, the peptide fragment identification list shows that: the 7 peptide fragments marked in the mass spectrum are compared with the full-length amino acid sequence of CILP2, and the peptide fragments are all in C-terminal fragments of the gene. The effect of CILP2 on heart senescence and myocardial hypertrophy was observed by injecting the C-terminus of CILP2 over-expressed by adeno-associated virus into senescent mice, demonstrating that the C-terminal fragment of CILP2 plays a major role in senescence.
According to the invention, a mouse aging model is established by naturally breeding 22-month wild-type mice, and 8-week-old young control wild-type C57BL/6 mice, 22-month-old aging control mice and 22-month-old aging CILP2 over-expression mice are used as experimental objects. Mouse heart ultrasonic detection, heart weighing, myocardial fibrosis, myocardial cell size detection, myocardial hypertrophy marker, myocardial aging marker and other phenotype detection are carried out. The results show that: compared with young mice, the aging control mice have obviously thickened wall thickness, aggravated myocardial fibrosis, increased myocardial cell cross-sectional area, obviously aggravated myocardial hypertrophy indexes and increased expression levels of aging markers P16, P21 and P53. Compared with an aging control group, the aging experimental group has the advantages that the over-expression of CILP2 can reduce the thickness of the wall of the heart chamber, reduce the weight of the heart, obviously reduce the size of the myocardial cells, reduce the index Myh6 of myocardial hypertrophy, obviously increase the index of Myh7, reduce the expression level of markers P16, P21 and P53 of myocardial aging, reduce the expression level of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in the myocardium and change the trend of the aging to young. The results show that the aging can promote the occurrence of the aging myocardial hypertrophy, the over-expression CILP2 can improve the aging myocardial hypertrophy by improving the aging marker gene expression, and theoretical basis and clinical basis are provided for researching a new target point and a new strategy for preventing, relieving and/or treating the aging myocardial hypertrophy.
In summary, the advantages and positive effects of the invention are:
the study of the present invention demonstrates that: in a model of aging, overexpression of CILP2 inhibited cardiac aging, reduced myocardial hypertrophy and fibrosis, and in particular, improved aging myocardial hypertrophy.
In conclusion, the invention discovers a new function of the CILP2 gene, namely the CILP2 has the functions of improving heart aging and myocardial hypertrophy; based on the functions of CILP2 in heart aging and myocardial hypertrophy, the method provides a target for developing a medicament for treating heart aging and myocardial hypertrophy; agonists of CILP2 may be used in the preparation of medicaments for the prevention, alleviation and/or treatment of heart aging and myocardial hypertrophy.
Drawings
FIG. 1 is a structural diagram of CILP2 gene;
FIG. 2 is a mouse treatment protocol;
FIG. 3 shows that CILP2 transcript levels in myocardium were upregulated after viral transfection. **: p < 0.01; ns: no statistical difference exists;
FIG. 4 is a heart ultrasonic testing mouse heart structure and function change, and mouse heart weight change; a: typical M-mode images of cardiac ultrasound; b: analyzing results of LVPWs, LVPWd and EF values of cardiac ultrasonic detection; c: mouse heart overall weight analysis, heart weight to weight/tibia length ratio analysis, left ventricle and right ventricle weight analysis. ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: no statistical difference exists;
FIG. 5 is HE staining and Wheat Germ Agglutinin (WGA) staining of myocardial tissue sections. A: performing HE staining on the cardiac tissue section, and performing 400-fold representative picture under a light microscope; b: WGA staining of cardiac tissue sections, 200-fold and 400-fold representative pictures under light microscopy; c: RT-PCR detects the change of Myh6 and Myh7 markers of cardiac hypertrophy at the mRNA level. *: p < 0.05; **: p < 0.01; ns: no statistical difference exists;
FIG. 6 shows the level changes of markers P16, P21 and P53 for myocardial aging and the level of mouse heart inflammation measured by RT-PCR. A: RT-PCR detects the level change of markers P16, P21 and P53 of myocardial aging; b: RT-PCR detects the level of heart inflammation in mice. *: p < 0.05; **: p < 0.01; ns: there were no statistical differences.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In the present invention, "about" means within 10%, preferably within 5% of a given value or range.
The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at 10 to 30 ℃, preferably 15 to 25 ℃.
The invention discloses application of CILP2 in preparing a medicament for improving heart aging and myocardial hypertrophy.
The experimental animal species, sex, size and source involved in the invention are: c57BL/6, male mice, young wild-type mice 8 weeks old, aged wild-type mice 22 months old. The experimental mice were purchased from Beijing Huafukang Biotech limited.
The C57BL/6 is taken as an experimental object, AAV9 virus is used for carrying out transcription level overexpression on CILP2 gene, and a heart aging and myocardial hypertrophy model is established by raising for 22 months, and the result shows that compared with an aging wild type control group, overexpression of CILP2 can obviously reduce the levels of P16, P21 and P53 which are indexes of heart aging of an aging mouse, reduce the levels of IL-1 beta, IL-6 and TNF-alpha which are indexes of myocardial inflammation factors of the aging mouse and Myh7 which is a marker of myocardial hypertrophy, increase the expression level of Myh6, reduce the wall thickness after the heart, weaken the fibrosis degree and reduce myocardial cells. The results prove that the expression level of CILP2 can regulate the occurrence of heart aging and myocardial hypertrophy, has protective effects on the heart aging and the myocardial hypertrophy, and can provide theoretical basis and clinical basis for researching new targets and new strategies for preventing and treating the heart aging and the myocardial hypertrophy. Therefore, against the above functions of CILP2, CILP2 can be used as a drug target for screening drugs for treating heart aging and myocardial hypertrophy.
CILP2 (human) referred to in this invention is numbered in NCBI databases: the gene ID is 148113, the nucleotide sequence number is NM-153221.2, and the protein sequence is NP-694953.2. Number of CILP2 (mouse) in NCBI database: the gene ID is 68709, the nucleotide sequence number NM-026818.2, and the protein sequence NP-081094. Sequences of mice used in the construction of adenoviruses.
The details of the present application are shown in the following examples.
Example application of CILP2 overexpression in aspects of improving heart aging, myocardial hypertrophy and the like of mice
1. Aging model construction and AAV9 virus transfection method
The mice used in the research institute are raised in the barrier environment of animal experiment centers in people hospitals of Wuhan university, the temperature in the barrier is constant (20-26 ℃), the humidity is constant (40-70%), the noise is low (less than 60 decibels), the ventilation frequency is more than 15 times/hour, the light/dark cycle is 12/12 hours, 4 animals are raised in each cage, and the mice freely take food. And naturally breeding to 22 months of age to construct an aging model.
The eukaryotic expression plasmid containing CILP 2C segment fragment (sequence shown in SEQ ID NO. 21) is constructed by using pHBAAV-CMV-MCS-3flag-T2A-ZsGreen plasmid vector by referring to CILP2 sequence of mouse by Hanhengcheng Biotech (Shanghai) Limited company, and the plasmid is packaged into AAV9-CILP2, while the unloaded pHBAAV-CMV-MCS-3flag-T2A-ZsGreen plasmid is packaged into AAV9-Neg as contrast. Mice were injected via tail vein with AAV9 virus at 16 months of age: the adenovirus AAV9-Neg of the control group and the adenovirus AAV9-CILP2 of the CILP2 overexpression group, the virus dose of each mouse injection is 5.5 multiplied by 1011particles, as shown in FIG. 2.
2. Detection method
Real-time fluorescent quantitative PCR (RT-PCR)
RNA extraction: adding 1ml of Trizol solution into the collected myocardial cells, repeatedly blowing on ice until the myocardial cells are fully lysed, standing for 5 minutes, adding 0.2ml of chloroform, violently shaking for 15-30 seconds, standing for 2-3 minutes, and centrifuging at 12000rpm x 15min at 4 ℃. The aqueous layer was pipetted into a fresh EP tube, 0.5ml isopropanol was added, the tube was gently mixed, allowed to stand at room temperature for 10 minutes, and centrifuged at 12000rpm x 10min at 4 ℃. The supernatant was discarded, 1ml of pre-cooled 75% ethanol was added to the pellet, the pellet was resuspended and washed thoroughly, and centrifuged at 12000rpm x 5min at 4 ℃. The supernatant was discarded, air dried, and an appropriate amount of DEPC water was added to promote RNA solubilization at 65 ℃. 2ul of RNA is taken for agarose gel electrophoresis to detect whether the RNA is successfully extracted, and the residual RNA is stored at the temperature of minus 80 ℃ when not used.
Real-time fluorescent quantitative PCR (RT-PCR): reverse transcription was performed using a reverse transcription kit from Thermo Fisher Scientific, using a Random primer as a primer, and reverse transcription was performed to cDNA. The reverse transcribed cDNA was diluted five times with ddH2O using SYBR from Thermo Fisher ScientificTMThe Green reagent detects the mRNA level, and the specific reaction system is shown in Table 1.
TABLE 1 real-time fluorescent quantitative PCR reaction System
Reaction conditions are as follows:
10min at 95 ℃; dissolution curves were plotted at 95 ℃ for 10sec, 60 ℃ for 10sec, 72 ℃ for 10sec, 40cycles, 40 ℃ for 30sec, and the final data were expressed as 2-△△CtAnd (6) carrying out analysis.
The sequences of the primers involved in the invention are shown in the following table:
3. identification of CILP2 overexpression at transcript level
Extracting total RNA from heart tissues of mice of each group, carrying out reverse transcription on the obtained cDNA, specifically detecting the mRNA expression level of CILP2 by adopting the method 2, and verifying whether the CILP2 can be specifically up-regulated by AAV9-CILP2 virus.
The results are shown in FIG. 3, the transcript level identifies that the transfected virus, and the level of cardiac myocyte CILP2 mRNA in the CILP2 overexpression group is increased by about 10 times compared with the level of control group CILP 2.
4. Ultrasound detection of changes in cardiac function and structure
Experiment was divided into 3 groups: the method comprises the following steps of carrying out detection on indexes such as an ejection function, ventricular septal thickness, ventricular posterior wall thickness and the like on hearts of mice of a Young 8-week-Old control group (Young control), an aging control group (Old-AAV9-Neg) and an aging CILP2 overexpression group (Old-AAV9-CILP2) on 3 groups of mice respectively adopting 2% isoflurane for anesthesia, then carrying out depilation on the chest parts of the mice, and carrying out detection on the indexes of the blood ejection function, the ventricular septal thickness, the ventricular posterior wall thickness and the like on the hearts of the mice by using an ultrasonic instrument.
The results are shown in FIG. 4: since the heart weight itself may be biased by body weight, the heart weight is corrected and analyzed by body weight and tibia length in this experiment. A: typical M-mode images of cardiac ultrasound; b: analyzing results of LVPWs, LVPWd and EF values of cardiac ultrasonic detection; c: mouse heart overall weight analysis, heart weight to weight/tibia length ratio analysis, left ventricle and right ventricle weight analysis. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: there were no statistical differences. The heart ultrasonic detection of the heart structure and the function of the mouse shows that the posterior wall thickness of the left ventricle of the aged mouse is obviously thickened, and the myocardial hypertrophy caused by aging is improved after the CILP2 is over-expressed, but the left ventricle ejection function of the mouse is not obviously influenced, which shows that the heart hypertrophy can be obviously improved by over-expressing CILP 2.
5. HE staining of myocardial tissue sections to observe myocardial morphological changes
After the myocardial tissue is fixed by 4% formaldehyde fixing solution, the myocardial tissue is embedded by paraffin to be used as a tissue section. HE staining method: paraffin section I xylene 2-5 minutes, II xylene 2-5 minutes, 100% ethanol 1min, 95% ethanol 1min, 80% ethanol 1min, 70% ethanol 1min, water washing with tap water and distilled water 0.5min, Harris sappan wood purple liquid 5 min. Hematoxylin violet is a basic dye that stains cell nuclei. The water was washed with tap water for 2 seconds, and the mixture was differentiated with 1% acidic alcohol (1mLHCl and 99mL of 70% alcohol) for 5 seconds, washed with tap water for a little while, and then added to a saturated aqueous solution of lithium carbonate to turn the slices blue, taking about 10 seconds in total. Then adding into 80% alcohol for 1min, and adding into 0.5% eosin alcohol solution for 1-2 min. Dehydrating with I95% ethanol for 2min, dehydrating with II 95% ethanol for 2min, dehydrating with I100% ethanol for 1min, dehydrating with II 100% ethanol for 1min, soaking in xylene carbolate mixture (1: 3) for transparence and absorbing water for 5 min. Then the carbolic acid is cleaned by I xylene for 1min and is made more transparent. And then carrying out dimethylbenzene II for 1min, taking out the slices from the dimethylbenzene, wiping off the dimethylbenzene except the slices by using dry gauze, immediately dripping a small drop of gum solution, and quickly taking a clean cover glass by using tweezers to cover the clean cover glass. As a result: the cell nucleus is bluish purple and the cytoplasm is red.
The morphological changes of the myocardial tissue were observed under a microscope. As shown in fig. 5A, the cardiomyocytes in the aged mice were significantly increased, and overexpression of CILP2 significantly reduced the cardiomyocyte size.
6. Wheat Germ Agglutinin (WGA) staining of myocardial tissue sections
1) Paraffin section dewaxing to water: placing the slices in xylene I15 min-xylene II 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-85% alcohol 5 min-75% alcohol 5 min-distilled water washing.
2) Dyeing: after the section is slightly dried, a circle is drawn around the tissue by a tissue pen, diluted WGA dye solution is dripped into the circle, and the section is incubated for 30min at 37 ℃ in a dark incubator.
3) DAPI counterstained nuclei: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, DAPI dye liquor is dripped into the circle, and the section is incubated for 10min at room temperature in a dark place.
4) Sealing sheet: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. The slices were slightly spun dry and mounted with an anti-fluorescent quenching mounting agent.
5) Taking a picture through microscopic examination: the sections were observed under a fluorescence microscope and images were collected. (DAPI ultraviolet excitation wavelength 330-380nm, emission wavelength 420nm, blue light emission; FITC excitation wavelength 465-495nm, emission wavelength 515-555nm, green light emission;
wheat germ agglutinin can specifically bind to myocardial cell membrane, and can stain myocardial cell membrane when they are combined, and can measure diameter, area, etc. by observing stained cells in myocardium under microscope, it can be analyzed whether myocardial cell is hypertrophic or not. As shown in fig. 5B, the cardiomyocytes in the aged mice were significantly increased, and overexpression of CILP2 significantly reduced the cardiomyocyte size.
7. Myocardial hypertrophy index detection
Total RNA is extracted from heart tissues of mice of each group, and the mRNA expression levels of the myocardial hypertrophy indexes Myh6 and Myh7 are detected by adopting a method in 2.
The results are shown in fig. 5C, the myo 6 expression level decreased in the aged mouse myocardium, the myo 7 expression level increased, and the upregulation of CILP2 expression partially reversed such changes.
8. Weighing the heart as a whole and the left and right ventricles respectively
After the mice are sacrificed by cervical dislocation, the heart is taken out and washed clean by PBS, and after the whole body is weighed, the left ventricle and the right ventricle are separated and then weighed separately. The weight of the heart of the mouse is detected, as shown in fig. 4C, after the intervention of the expression level of CILP2, the weights of the left ventricle and the right ventricle of the heart are reduced, and the conclusion obtained after the correction of the weight and the length of the tibia is consistent, which indicates that the heart hypertrophy can be remarkably improved by over-expression of CILP 2.
9. Detection of aging and inflammation indicators
Total RNA was extracted from heart tissue of mice in each group, and mRNA expression levels of senescence-associated factors P16, P21 and P53 and mRNA expression levels of inflammatory factors IL-1 β, IL-6 and TNF- α were measured by the method of 2.
The results are shown in FIG. 6. P16, P21 and P53 are indicators of heart aging, the expression levels of the three genes in the myocardium are increased along with the increase of the age, and the expression levels of P16, P21 and P53 in the myocardium are reversed after the CILP2 is up-regulated. In addition, since chronic inflammation is a characteristic and pathogenic factor of aging, the expression levels of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in the myocardium of three groups of mice are also tested, and the level of inflammatory factors in the myocardium of aged mice is obviously increased, while the level of inflammatory factors in the myocardium is reduced after the expression of CILP2 is up-regulated. Although not statistically significant, it was trend-finding that upregulation of CILP2 expression might improve heart aging.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
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