Polypeptide and application thereof in preparation of related products for inhibiting myocardial remodeling

Abstract

The invention relates to a polypeptide and application thereof in preparation of related products for inhibiting myocardial remodeling. The CTRP9 229-333 polypeptide can improve the cardiac function of a patient with cardiac diseases, so that the CTRP9 229-333 polypeptide can be used for treating myocardial remodeling diseases caused by stable angina pectoris, unstable angina pectoris, myocardial infarction, chronic ischemic heart disease, coronary spasmodic heart disease, myocarditis and the like. Besides, the CTRP9 229-333 polypeptide not only can inhibit cardiac fibrosis, but also can inhibit myocardial hypertrophy, and the myocardial cell apoptosis inhibiting capacity of the CTRP9 229-333 polypeptide is higher than that of the full-length protein CTRP9. Compared with the full-length protein CTRP9, the CTRP9 229-333 polypeptide has remarkable advantages in the aspects of absorption efficiency, biological activity, nutritional value, safety, applicability and the like compared with the protein. Due to the advantages, the polypeptide has wide application prospects in the fields of nutritional supplement, health care, disease treatment and the like.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine technology, and in particular to a polypeptide. The present disclosure also relates to the use of the polypeptide in preparing products related to inhibiting myocardial remodeling. Background Art

[0002] Myocardial remodeling is a process in which the body initiates its own attempt to repair and compensate. When the heart is damaged or in a hemodynamic stress response, changes in molecular and gene expression lead to heart deformation, heart enlargement, and decreased ejection fraction, leading to heart failure. Myocardial remodeling often occurs after myocardial infarction, or in patients with chronic myocarditis, dilated cardiomyopathy, heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, etc. After the disease is initiated, the heart will remodel in order to save itself. It can be seen that early inhibition of myocardial remodeling is an effective strategy for delaying the occurrence of heart failure in patients with cardiovascular disease.

[0003] Cardiovascular myocardial remodeling refers to the adaptive changes in cardiac structure and function under various harmful stimuli. A large number of experimental data show that during myocardial remodeling, myocardial cell loss caused by pathological pathways such as necrosis, apoptosis or autophagy; myocardial fibrosis caused by fibroblast proliferation and extracellular matrix (ECM) reorganization; and myocardial hypertrophy caused by reduced cardiac contractility due to mitochondrial dysfunction and metabolic abnormalities may be the main pathological mechanisms of myocardial remodeling. Myocardial remodeling is a change in cardiac structure and function caused by cardiovascular disease. It is a significant pathological manifestation and an important pathological basis for the prognosis and development of cardiovascular disease.

[0004] At present, although there are many drugs that can inhibit myocardial remodeling in clinical practice, such as angiotensin-converting enzyme inhibitors, beta-blockers, etc., the control rate of myocardial remodeling in cardiovascular diseases is still poor, and the incidence of cardiovascular diseases is increasing year by year. Therefore, early search for targets to prevent or intervene in this process and reverse myocardial remodeling in cardiovascular diseases is of great significance for reducing the incidence of cardiovascular diseases, improving patient prognosis, reducing patient mortality, and reducing national medical costs.

[0005] The C1q / TNF related protein family (CTRP) consists of a group of highly conserved proteins with similar structures to adiponectin. They belong to the C1q family of small secreted proteins and include 15 members. Among all the CTRP family members, the globular domain of CTRP9 has the most amino acid overlap sequences with adiponectin, and CTRP9 is the only CTRP member that can bind to adiponectin to form a hybrid oligomer. CTRP9 is a secreted protein that consists of a signal peptide, a short amino terminus, a variable number of Gly-XY collagen repeats, and a globular domain C1q carboxyl terminus (gCTRP9). Studies on the expression and distribution of CTRP9 have revealed that the heart is the third most expressed organ for CTRP9, and the mRNA level of CTRP9 in heart tissue is 2.5 times that of adipose tissue. CTRP9 is also the most expressed adipokine in the heart (its expression level is 100 times that of adiponectin and 1.6 times that of CTRP9 protein in plasma).

[0006] Studies on the biological functions of CTRP9 have shown that CTRP9 is a potential cardioprotective molecule that can attenuate acute ischemia-reperfusion injury, reduce ventricular remodeling caused by myocardial infarction and immune dysfunction, and slow down ischemic heart failure in animals. In a mouse model of sleep apnea-hypopnea syndrome (OSA) combined with ischemic myocardial injury, the expression level of CTRP9 in myocardial tissue was significantly reduced, and exogenous administration of CTRP9 can effectively reduce the severity of myocardial remodeling in mice. This suggests that CTRP9 produced by the heart plays an important regulatory role in the process of myocardial injury and protection. Therefore, as a potential cardioprotective factor, CTRP9 has become a hot research topic in the early intervention of the occurrence and development of cardiovascular disease.

[0007] Studies have found that CTRP9 can inhibit myocardial cell apoptosis, improve myocardial fibrosis, and promote cardiac repair, which can have a positive effect on myocardial remodeling in many ways, but CTRP9 has no significant effect on myocardial hypertrophy. Other studies have shown that the heart can cut exogenous full-length CTRP9 into gCTRP9, which is an important post-transcriptional modification product for CTRP9 to exert its biological effects. It has biological activity and can activate key protein kinases in myocardial cells, effectively alleviating ischemia-reperfusion injury. It can be seen that CTRP9 protease cleavage is a physiologically relevant and biologically important form of post-translational modification. Summary of the invention

[0008] In order to solve the problems existing in the prior art, the present disclosure provides a polypeptide and its application in preparing products related to inhibiting myocardial remodeling.

[0009] According to the first aspect of the present disclosure, a polypeptide is provided, wherein the amino acid sequence of the polypeptide is:

[0010] NEFNHYDTAAGKFTCHIAGVYYFTYHITVFSRNVQVSLVKNGVKILHTKDAYMSSEDQASGGIVLQLKLGDEVWLQVTGGERFNGLFADEDDDTTFTGFLLFSSP.

[0011] In one embodiment of the present disclosure, the polypeptide is obtained by the following method:

[0012] Step a: co-incubate the recombinant protein CTRP9 with Cathepsin G protein to obtain the experimental group sample; mix the recombinant protein CTRP9 with physiological saline to obtain the control group sample;

[0013] Step b: Perform quantitative electrophoresis on the two groups of samples, compare the difference bands between the two groups of samples after staining, and cut the difference band near the molecular weight of 17KDa in the experimental group;

[0014] Step c: performing mass spectrometry detection on the differential bands of the experimental group to obtain the amino acid sequence of the polypeptide;

[0015] Step d: synthesizing the polypeptide based on the amino acid sequence. According to the second aspect of the present disclosure, there is also provided a use of the polypeptide according to the first aspect of the present disclosure in preparing a product related to inhibiting myocardial remodeling.

[0016] In one embodiment of the present disclosure, the related products are used to treat myocardial remodeling-related diseases, which at least include: stable angina pectoris, unstable angina pectoris, myocardial infarction, chronic ischemic heart disease, coronary artery spasm heart disease, and myocarditis.

[0017] In one embodiment of the present disclosure, the myocardial remodeling-related diseases also include: diabetes combined with myocardial ischemia-reperfusion.

[0018] In one embodiment of the present disclosure, the myocardial remodeling-related diseases further include: heart failure with preserved ejection fraction.

[0019] In one embodiment of the present disclosure, the related product is used to inhibit myocardial hypertrophy and to inhibit cardiac fibrosis.

[0020] In one embodiment of the present disclosure, the related product is used to inhibit cardiomyocyte apoptosis.

[0021] In one embodiment of the present disclosure, the related product is a drug or a pharmaceutical composition; the drug or pharmaceutical composition also includes a diluent, an adjuvant and / or a carrier that can be used in the pharmaceutical field.

[0022] In one embodiment of the present disclosure, the related product is food, health care product, enteral nutrition preparation, veterinary drug or feed additive.

[0023] One beneficial effect of the present disclosure is that a polypeptide CTRP9229-333 capable of inhibiting myocardial remodeling is provided, which can improve the cardiac function of patients with heart diseases, and thus can be used to treat diseases that cause myocardial remodeling, such as stable angina pectoris, unstable angina pectoris, myocardial infarction, chronic ischemic heart disease, coronary artery spasm heart disease, myocarditis, etc. Specifically, the CTRP9 229-333 polypeptide can at least improve the cardiac function of diabetic mice with myocardial ischemia-reperfusion (HFD+IR) model mice and heart failure with preserved ejection fraction (HFpEF) model mice, and it can inhibit myocardial remodeling by inhibiting the expression of cardiac fibrosis genes and hypertrophy genes.

[0024] In addition, although the full-length protein CTRP9 can improve myocardial fibrosis, it has no significant effect on myocardial hypertrophy, while the CTRP9 229-333 polypeptide can not only inhibit cardiac fibrosis, but also inhibit myocardial hypertrophy, and the ability of CTRP9229-333 polypeptide to inhibit myocardial cell apoptosis is stronger than that of the full-length protein CTRP9. Compared with the full-length protein CTRP9, the CTRP9 229-333 polypeptide has significant advantages over the protein in terms of absorption efficiency, biological activity, nutritional value, safety and applicability. These advantages make the polypeptide have broad application prospects in the fields of nutritional supplementation, health care and disease treatment.

[0025] Other features and advantages of the present disclosure will become apparent from the following detailed description of exemplary embodiments of the present disclosure with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0027] Figure 1 This is a diagram of the electrophoresis experiment results in Example 1 of the present disclosure.

[0028] Figure 2 This is a diagram of mass spectrometry detection results in Example 1 of the present disclosure.

[0029] Figure 3 This is a diagram of the results of the cardiomyocyte apoptosis experiment in Example 2 of the present disclosure.

[0030] Figure 4 It is a schematic diagram of the HFD+IR model construction process in the third embodiment of the present disclosure.

[0031] Figure 5 This is a diagram of the anatomical measurement experiment results of the HFD+IR model in Example 3 of the present disclosure.

[0032] Figure 6 This is a diagram of the ultrasonic experiment results of the HFD+IR model in Example 3 of the present disclosure.

[0033] Figure 7 This is a data result diagram of ejection fraction and short-axis shortening rate of the HFD+IR model ultrasound experiment in Example 3 of the present disclosure.

[0034] Figure 8 This is a diagram of the HFD+IR model PET-CT experimental results in Example 3 of the present disclosure.

[0035] Fig. 9 This is a result diagram of the SUV value data of the HFD+IR model PET-CT experiment in Example 3 of the present disclosure.

[0036] Fig.10 This is a diagram of the Vevo strain analysis experimental results of the HFD+IR model in Example 3 of the present disclosure.

[0037] Fig.11 This is a diagram of the experimental data results of Vevo strain analysis using the HFD+IR model in Example 3 of the present disclosure.

[0038] Fig.12 This is a diagram of the results of the masson staining experiment of the HFD+IR model in Example 3 of the present disclosure.

[0039] Fig.13 This is a graph showing the WGA staining experiment results of the HFD+IR model in Example 3 of the present disclosure.

[0040] Fig.14 This is a graph showing the experimental results of qRT-PCR verification of cardiac fibrosis gene levels in the HFD+IR model in Example 3 of the present disclosure.

[0041] Fig.15 This is a graph showing the experimental results of qRT-PCR verification of cardiac hypertrophy gene levels in the HFD+IR model in Example 3 of the present disclosure.

[0042] Fig.16 It is a schematic diagram of the HFpEF model construction process in the fourth embodiment of the present disclosure.

[0043] Fig.17 This is a diagram of the anatomical measurement experimental results of the HFpEF model in Example 4 of the present disclosure.

[0044] Fig.18 This is a graph showing the results of the exercise capacity and blood glucose measurement experiment of HFpEF model mice in Example 4 of the present disclosure.

[0045] Fig.19 This is a diagram of the ultrasonic experiment results of the HFpEF model in Example 4 of the present disclosure.

[0046] Fig. 20 This is a diagram of cardiac diastolic data results of an ultrasound experiment of a HFpEF model in the fourth embodiment of the present disclosure.

[0047] Fig.21 It is a diagram of the Vevo strain analysis experimental results of the HFpEF model in the fourth embodiment of the present disclosure.

[0048] Fig. 22 This is a graph showing the overall longitudinal strain data results of the Vevo strain analysis experiment of the HFpEF model in the fourth embodiment of the present disclosure.

[0049] Fig.23 This is a diagram showing the results of a Masson staining experiment on the HFpEF model in Example 4 of the present disclosure.

[0050] Fig.24 This is a graph showing the results of a WGA staining experiment on a HFpEF model in Example 4 of the present disclosure.

[0051] Fig.25 This is a graph showing the experimental results of qRT-PCR verification of cardiac fibrosis gene levels in the HFpEF model in Example 4 of the present disclosure.

[0052] Fig.26 This is a graph showing the experimental results of qRT-PCR verification of cardiac hypertrophy gene levels in the HFpEF model in Example 4 of the present disclosure. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present disclosure unless otherwise specifically stated.

[0054] The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the present disclosure, its application, or uses.

[0055] Technologies, methods, and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered as part of the specification.

[0056] It should be noted that like reference numerals and letters refer to similar items in the following figures, and therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0057] In this document, “upper”, “lower”, “front”, “back”, “left”, “right”, etc. are only used to indicate the relative position relationship between related parts, rather than to limit the absolute positions of these related parts.

[0058] In this article, "first", "second", etc. are only used to distinguish each other, and do not indicate the importance and order, or the premise of each other's existence.

[0059] In this document, “equal”, “same”, etc. are not strictly limited in a mathematical and / or geometric sense, but also include errors that can be understood by those skilled in the art and are allowed in manufacturing or use.

[0060] The present disclosure provides a polypeptide and its application in preparing products related to inhibiting myocardial remodeling, wherein the amino acid sequence of the polypeptide is:

[0061] NEFNHYDTAAGKFTCHIAGVYYFTYHITVFSRNVQVSLVKNGVKILHTKDAYMSSEDQASGGIVLQLKLGDEVWLQVTGGERFNGLFADEDDDTTFTGFLLFSSP.

[0062] The polypeptide provided by the present disclosure is CTRP9 229-333, which is enzymatically cleaved from the recombinant protein CTRP9. In a specific embodiment of the present disclosure, the polypeptide can be obtained by the following method:

[0063] Step a: co-incubate the recombinant protein CTRP9 with the Cathepsin G protein to obtain the experimental group sample; mix the recombinant protein CTRP9 with physiological saline to obtain the control group sample. Specifically, Cathepsin G protein is an enzyme that can specifically cut the CTRP9 protein. In the experimental group, the recombinant protein CTRP9 is co-incubated with the Cathepsin G protein, and the Cathepsin G protein can enzymatically cut the 229th site of the recombinant protein CTRP9 to obtain the experimental group sample containing the target polypeptide. The control group sample contains the full-length CTRP9 protein, so it can be used as a control in the subsequent electrophoresis step.

[0064] Step b: Perform quantitative electrophoresis on the two groups of samples, compare the difference bands between the two groups of samples after staining, and cut the difference band near the molecular weight of 17KDa in the experimental group. Specifically, observe whether there are difference bands between the two groups after Coomassie Brilliant Blue staining. If no difference bands are found, it means that the experiment has failed and needs to be repeated. If difference bands are found, the bands are cut according to the observed difference bands. According to the position of the protein marker, compared with the control group, the target protein band near the molecular weight of 17KDa in the experimental group is cut, and a band containing at least part of the target polypeptide has been obtained.

[0065] Step c: Perform mass spectrometry on the differential bands of the experimental group to obtain the amino acid sequence of the polypeptide. Specifically, add trypsin to the target band and perform enzymatic hydrolysis for 12-16 hours; put the enzymatically hydrolyzed sample into an ultrasonic instrument for 15-20 minutes, aspirate the peptides, vacuum dry, dissolve the peptides with sample dissolving solution (0.1% formic acid, 2% acetonitrile), vortex thoroughly, and then centrifuge at 13000rpm, 4℃ for 10 minutes. The supernatant obtained by centrifugation is transferred to the sample tube and enters the mass spectrometer for online detection. After data analysis, the amino acid sequence is obtained.

[0066] Step d: Synthesizing a polypeptide based on the amino acid sequence. Specifically, a high-purity target polypeptide can be further synthesized based on the amino acid sequence obtained in step c, so that it can be used in experiments.

[0067] The peptide CTRP9 229-333 can inhibit myocardial remodeling and improve the cardiac function of patients with heart disease, thereby being used to treat myocardial remodeling-related diseases, such as some diseases that easily cause myocardial remodeling. The related diseases at least include: stable angina pectoris, unstable angina pectoris, myocardial infarction, chronic ischemic heart disease, coronary artery spasm heart disease, myocarditis, etc. More specifically, the related diseases also include: diabetes combined with myocardial ischemia-reperfusion and heart failure with preserved ejection fraction.

[0068] The present invention discloses the establishment of diabetic mice with myocardial ischemia-reperfusion (HFD+IR) model and HFpEF model, and proves through experiments that CTRP9 229-333 polypeptide can at least improve the cardiac function of diabetic mice with myocardial ischemia-reperfusion (HFD+IR) model and HFpEF model. It can be seen that CTRP9 229-333 polypeptide has a significant therapeutic effect on at least two diseases, diabetic mice with myocardial ischemia-reperfusion and HFpEF.

[0069] In one embodiment of the present disclosure, the related products are used to inhibit myocardial hypertrophy and to inhibit cardiac fibrosis. Specifically, the present disclosure verifies through experiments that the related products containing CTRP9 229-333 polypeptide can inhibit myocardial remodeling by inhibiting the expression of cardiac fibrosis genes and hypertrophy genes. Although the full-length protein CTRP9 in the prior art can improve myocardial fibrosis, it has no significant effect on myocardial hypertrophy, while the CTRP9229-333 polypeptide disclosed in the present disclosure can not only inhibit cardiac fibrosis, but also inhibit myocardial hypertrophy.

[0070] In one embodiment of the present disclosure, the related products are used to inhibit cardiomyocyte apoptosis. Specifically, the present disclosure verifies through experiments that the related products containing CTRP9 229-333 polypeptide can inhibit cardiomyocyte apoptosis, and the ability of CTRP9229-333 polypeptide to inhibit cardiomyocyte apoptosis is stronger than that of the full-length protein CTRP9.

[0071] In addition, compared with the full-length protein CTRP9, the CTRP9 229-333 peptide has significant advantages over the protein in terms of absorption efficiency, biological activity, nutritional value, safety and applicability. These advantages make the peptide have broad application prospects in the fields of nutritional supplementation, health care and disease treatment.

[0072] In one embodiment of the present disclosure, the related product is a drug or a pharmaceutical composition. The CTRP9 229-333 polypeptide can be used in combination with other drugs for treating or alleviating diseases related to myocardial remodeling in related products. Although the present disclosure does not provide relevant experiments for the combined use with other drugs for treating or alleviating diseases related to myocardial remodeling, for those skilled in the art, the CTRP9 229-333 polypeptide, as a polypeptide capable of inhibiting myocardial remodeling, can be used in combination with other drugs to achieve the therapeutic purpose of the present disclosure.

[0073] Specifically, the related products of the present disclosure may be pharmaceutical compounds, pharmaceutical compositions, etc., or may be drugs in various dosage forms such as tablets, granules, powders, capsules, injections, etc. For example, the related products of the present disclosure may be injection preparations, oral tablets, or oral capsules. In addition, the drug or pharmaceutical composition also includes a pharmaceutically acceptable excipient; a pharmaceutically acceptable excipient refers to any diluent, adjuvant, and / or carrier that can be used in the pharmaceutical field. On the basis that the CTRP9229-333 polypeptide disclosed in the present disclosure can treat myocardial remodeling-related diseases, it is a conventional choice for those skilled in the art to reasonably select suitable diluents, adjuvants, and / or carriers, which will not be described in detail here.

[0074] In one embodiment of the present disclosure, the related product is a food, a health product, an enteral nutrition preparation, a veterinary drug or a feed additive. Further, the food, the health product, the enteral nutrition preparation, the veterinary drug or the feed additive also contains conventional adjuvants. Reasonable selection of suitable conventional adjuvants is a conventional choice for those skilled in the art and is not described in detail here. For example, conventional adjuvants may include: one or more of fillers, flavoring agents, adhesives, disintegrants, lubricants, antacids, and nutritional enhancers.

[0075] Example 1: Acquisition and identification of CTRP9 229-333 polypeptide

[0076] Cathepsin G is an enzyme that can specifically cleave CTRP9 protein, and the cleavage product is CTRP9 229-333 polypeptide. Based on this, this example uses specific enzyme cleavage to obtain CTRP9 229-333 polypeptide, and performs sequencing analysis on it to obtain the sequence of the polypeptide.

[0077] 1. Description of experimental materials

[0078] 1.1. Main reagents: recombinant protein CTRP9 (purchased from R&D Systems, USA), Cathepsin G protein (purchased from Abcam, UK), conventional Coomassie Brilliant Blue staining solution (purchased from Regen Biotech Co., Ltd.), Coomassie Brilliant Blue decolorizing solution (purchased from Beyotime Biotechnology Co., Ltd.), protein marker (purchased from Thermo Fisher Scientific, USA).

[0079] 1.2. Main instruments and equipment: electrophoresis instrument (purchased from Bio-Rad, USA).

[0080] 2. Experimental methods

[0081] The experiment was divided into two groups: CTRP9+normal saline group and CTRP9+Cathepsin G group. In the CTRP9+Cathepsin G group, the recombinant protein CTRP9 and Cathepsin G protein were incubated at 37°C for 1 hour to obtain peptides of different molecular weights by enzyme digestion.

[0082] Subsequently, the two groups of samples were subjected to quantitative electrophoresis, and after Coomassie brilliant blue staining, the difference bands between the two groups were observed, and the bands were cut according to the observed difference bands. The effective fragment of CTRP9 protein that exerts myocardial protection is the globular CTRP9 molecule (molecular weight of 16kd) located at the C-terminus, which indicates that the active domain of CTRP9 may be a low molecular weight polypeptide. In this embodiment, in order to clarify the specific site of Cathepsin G cutting CTRP9 and the resulting polypeptide fragments, Cathepsin G cuts the full-length CTRP9 to produce multiple fragments (i.e., in the multiple difference bands compared with the CTRP9 + saline group), select the difference band fragment with a lower molecular weight (about 17kd) for mass spectrometry detection, in the hope of screening out the CTRP9 low molecular weight polypeptide with a protective effect.

[0083] Mass spectrometry detection steps:

[0084] Trypsin was added to the target band and enzymatically digested for 12-16 hours; the enzymatically digested sample was placed in an ultrasonic instrument for 15-20 minutes, the peptides were aspirated, vacuum-dried, and the peptides were dissolved in a sample dissolving solution (0.1% formic acid, 2% acetonitrile), fully vortexed, and then centrifuged at 13000rpm, 4℃ for 10 minutes. The supernatant obtained by centrifugation was transferred to a sample tube and entered into a mass spectrometer Thermo Scientific Q Exactive for online detection; the original file obtained after mass spectrometry detection was processed and converted by MM FileConversion software to obtain an MGF format file, and then MASCOT was used to search the NCBI Homo sapiens database and the uniprot Homo sapiens database for data analysis. The CTRP9 229-333 polypeptide sequence was found by blast sequence analysis.

[0085] 3. Experimental results

[0086] The electrophoresis results are as follows Figure 1 As shown in the figure, the electrophoresis experiment results are explained: the leftmost column is the marker, the middle column is the full-length CTRP9 protein without enzyme digestion, and the molecular weight of the CTRP9 protein is between 34-43KD; the right column is the product after co-incubation of CTRP9+CathepsinG, and the differential band circled in the box in the figure corresponds to a molecular weight of about 17KD.

[0087] Will Figure 1 The band in the box was cut out and subjected to mass spectrometry detection. The mass spectrometry detection results were as follows: Figure 2 As shown, the CTRP9 229-333 polypeptide sequence was found by blast sequence analysis:

[0088] (229)NEFNHYDTAAGKFTCHIAGVYYFTYHITVFSRNVQVSLVKNGVKILHTKDAYMSSEDQASGGIVLQLKLGDEVWLQVTGGERFNGLFADEDDDTTFTGFLLFSSP(333)

[0089] In subsequent examples, CTRP9 229-333 polypeptide was synthesized based on the above sequence (synthesized by Jimeijiaer Biotechnology Co., Ltd.).

[0090] Example 2: Myocardial cell apoptosis experiment

[0091] 1. Experimental Materials

[0092] The CTRP9 229-333 polypeptide synthesized based on the sequencing results in Example 1 (synthesized by Jimeijiaer Biotechnology Co., Ltd.); recombinant protein CTRP9 (purchased from R&D Systems, USA); HBSS buffer (purchased from Beijing Solebow Technology Co., Ltd.); DMEM and L-15 cell culture medium (purchased from Gibco, USA); 0.25% trypsin (purchased from Gibco, USA, and the different concentrations of trypsin used in this example and subsequent examples are all derived from this concentration dilution); PBS buffer (purchased from Beijing Solebow Technology Co., Ltd.); fetal bovine serum (purchased from Gibco, USA); 2.5L cell anoxic culture gas production bag (purchased from Mitsubishi, Japan); 2.5L anaerobic sealed culture box (purchased from Mitsubishi, Japan); type II collagenase (purchased from Worthington Biological Company, USA); neonatal rat cardiomyocytes (from SD rats); cell culture incubator (purchased from Thermo Fisher Scientific, USA); Annxin V-FITC / PI cell apoptosis detection kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.); flow tube (purchased from BD Falcon, USA); flow cytometer (purchased from BD, USA); constant temperature shaker (purchased from Thermo Fisher Scientific, USA).

[0093] 2. Experimental methods

[0094] Twenty SD rat neonates were purchased and neonate cardiomyocytes were isolated by the following method.

[0095] 1) Soak the rat in 75% alcohol, surgically remove the living heart, and immediately place the heart in a centrifuge tube containing 40 ml HBSS buffer to cool and wash.

[0096] 2) After taking out all the rat hearts, rotate the centrifuge tube to wash the hearts, discard the above washing solution, and wash the hearts 3 times with HBSS buffer.

[0097] 3) Move the heart into a 100 mm culture dish. Place the heart tissue on ice and use small curved scissors to cut the heart into pieces smaller than 1 mm. 3 size.

[0098] 4) Add new HBSS solution to the culture dish and adjust the final volume to about 9 ml.

[0099] 5) Add 2.25 ml of trypsin to the culture dish to adjust the final concentration to 0.05% (the trypsin concentration used is 0.25%).

[0100] 6) The culture dish was then placed in a 4°C refrigerator overnight (approximately 16 hours).

[0101] 7) Transfer the myocardial tissue and buffer that have been refrigerated overnight into a 50 ml centrifuge tube, add 6 ml of 10% fetal bovine serum, and mix well by pipetting.

[0102] 8) Preheat the myocardial tissue and buffer in a sterile water bath at 37°C for 30 minutes, and the final volume of the solution is 14 ml. Note: Do not add culture medium containing calcium ions before heating the myocardial tissue.

[0103] 9) Add 30 mg of type II collagenase (the final concentration of type II collagenase is 0.1% to 0.2%) into the centrifuge tube, mix carefully, and place the centrifuge tube in a constant temperature shaker at 37°C, at a speed of 170g for 30 min.

[0104] 10) Add 6 ml of L-15 culture medium and gently blow the cells to separate the cells from the cells and tissues.

[0105] 11) Let the solution settle for 4 minutes, and filter about 15 ml of the liquid through a 70 μm filter into a new centrifuge tube.

[0106] 12) Continue digestion for 20 min in a constant temperature shaker, add 5 ml of L-15 medium, and gently blow the cells to separate the cells from the cells and tissues; filter the liquid with a 70 μm filter into a new centrifuge tube; depending on the digestion situation, the above digestion steps can be repeated once more.

[0107] 13) Divide the solution into three 15 ml centrifuge tubes to make the volumes equal for easy centrifugation. Finally, let the filtered cells settle naturally at room temperature for 15 minutes to allow the partially degraded collagenase to be completely digested.

[0108] 14) After sedimentation is complete, place the centrifuge tube in a centrifuge and centrifuge at 100g for 5 minutes.

[0109] 15) Discard the supernatant, add 10 ml of 15% fetal bovine serum + DMEM culture medium to each centrifuge tube for resuspending, and plate the cells in three 6-well plates in a 37°C incubator for differential attachment for 60 minutes.

[0110] 16) After 60 minutes, collect the culture medium containing cardiomyocytes and place it in a centrifuge tube.

[0111] 17) Rinse the 6-well plate 5 times with PBS to confirm that there are no suspended cells, then add 10 ml of 10% FBS + DMEM culture medium to each well and continue culturing.

[0112] 18) Spread the culture medium containing cardiomyocytes into two 6-well plates, 2 ml per well. Observe the cell adhesion and growth after 24 hours; observe for contamination after 6-8 hours, and change the medium after 48 hours.

[0113] After isolating neonatal rat cardiomyocytes using the above steps, they were divided into the following three groups:

[0114] Control group: physiological saline was added to the culture medium for 24 hours;

[0115] CTRP9 group: CTRP9 full-length protein was added to the culture medium for stimulation for 24 hours;

[0116] CTRP9 229-333 group: CTRP9 229-333 peptide was added to the culture medium for stimulation for 24 hours.

[0117] Seven hours after the stimulants were added to the three groups of myocardial cells, the three groups of neonatal rat myocardial cells were placed in a 2.5L anaerobic sealed culture box, and a 2.5L cell hypoxia culture gas production bag was placed, the culture box was sealed, and placed in a 37°C, 5% CO2 cell culture incubator for 6 hours to perform cell hypoxia treatment. The three groups of neonatal rat myocardial cells were then taken out of the anaerobic sealed culture box and continued to be placed in a 37°C, 5% CO2 cell culture incubator for 12 hours of reoxygenation treatment, thereby simulating the phenomenon of myocardial cell ischemia-reperfusion.

[0118] After the hypoxia / reoxygenation treatment, the three groups of cells were collected and pretreated with the Annxin V-FITC / PI cell apoptosis detection kit, and then the apoptosis of the three groups of cardiomyocytes was detected by flow cytometry. The specific operation steps are as follows:

[0119] Annxin V-FITC / PI Cell Apoptosis Detection Kit contains the following 1× Binding Buffer, Annexin V-FITC and PI Staining Solution reagents.

[0120] The sample staining steps are as follows:

[0121] 1) Digest the three groups of neonatal mouse cells with 0.05% trypsin and collect the cells by centrifugation at 300g and 4°C for 5 min. Trypsin digestion should not be too long to avoid false positives.

[0122] 2) Wash the cells twice with pre-cooled PBS, centrifuging at 300g for 5 min at 4°C each time. Collect 1-5×10 5 cell.

[0123] 3) Aspirate and discard PBS, and add 100 μL 1× Binding Buffer to resuspend the cells.

[0124] 4) Add 5 μL Annexin V-FITC and 10 μL PI Staining Solution and mix gently.

[0125] 5) Incubate at room temperature away from light for 10-15 minutes.

[0126] 6) Add 400 μL 1× Binding Buffer, mix well and place on ice. Detect the sample by flow cytometry or fluorescence microscope within 1 hour.

[0127] The flow cytometric analysis (BD: FACSCanto II) method is as follows:

[0128] The maximum excitation wavelength of FITC is 488 nm, the maximum emission wavelength is 525 nm, and the green fluorescence of FITC is detected in the FL1 channel; the maximum excitation wavelength of the PI-DNA complex is 535 nm, the maximum emission wavelength is 615 nm, and the red fluorescence of PI is detected in the FL2 or FL3 channel. FlowJo software was used for analysis and a scatter plot was drawn with FITC as the horizontal axis and PI as the vertical axis, and the scatter plot was divided into four quadrants. In a typical experiment, cells can be divided into three subpopulations, living cells have only very low-intensity background fluorescence, early apoptotic cells have only strong green fluorescence, and late apoptotic cells have dual staining of green and red fluorescence. That is, the third quadrant shown in the scatter plot is normal living cells, the second quadrant is early apoptotic cells, and the fourth quadrant is late apoptotic cells. The proportion of cells in the second quadrant is taken as the percentage of cell apoptosis.

[0129] 3. Experimental results

[0130] like Figure 3 As shown, after hypoxia / reoxygenation treatment, there were nearly 80% late apoptotic cells in the control group. Compared with the control group, both the CTRP9 full-length protein and the CTRP9 229-333 polypeptide significantly reduced the apoptosis level of hypoxia / reoxygenation cardiomyocytes; further, compared with the CTRP9 full-length protein, the CTRP9 229-333 polypeptide had a stronger ability to inhibit cardiomyocyte apoptosis.

[0131] Example 3: Effect of CTRP9 229-333 polypeptide in the diabetes combined with myocardial ischemia-reperfusion model (HFD+IR)

[0132] Clinically, diabetic patients often have cardiovascular and cerebrovascular diseases. Long-term chronic hyperglycemia in diabetes will stimulate the blood vessel wall after the blood sugar level rises, and then cause certain damage to the blood vessel wall; damage can easily lead to atherosclerosis, which in turn leads to stenosis and occlusion of blood vessels. In the long run, it will lead to stenosis and occlusion of blood vessels, thereby causing cardiovascular and cerebrovascular diseases. In addition, diabetic patients often have other metabolic diseases, such as hyperlipidemia, hyperuricemia, and hypertension, and these metabolic diseases are also important risk factors for the occurrence of cardiovascular and cerebrovascular diseases. Diabetes and these metabolic diseases complement each other and jointly lead to the occurrence of cardiovascular and cerebrovascular diseases. Myocardial remodeling is prone to occur in patients with diabetes + cardiovascular disease. This example establishes a diabetes combined with myocardial ischemia reperfusion (HFD+IR) model to verify the therapeutic effect of CTRP9 229-333 polypeptide on myocardial remodeling.

[0133] Ischemia-Reperfusion injury (IRI) refers to a pathological phenomenon in which a tissue or organ is in an ischemic state due to insufficient blood supply, and is severely damaged when it regains blood supply. IRI often occurs after heart-related surgery and is called myocardial ischemic reperfusion injury (MI / R). MI / R may occur in myocardial infarction treatments such as coronary angioplasty, coronary artery bypass grafting (CABG), thrombolytic therapy or primary percutaneous coronary intervention (PPCI). MI / R is a key factor affecting patient prognosis, increasing infarct size and promoting long-term adverse cardiac remodeling, and ultimately leads to myocardial damage. In this example, the myocardial damage of mice was caused by establishing an ischemia-reperfusion model, thereby verifying the protective effect of CTRP9 229-333 polypeptide on myocardial damage and the therapeutic effect on myocardial remodeling.

[0134] 1. Experimental animals: Wild-type mice (purchased from Beijing Huafukang Experimental Animal Company, strain C57BL / 6J, male, 8 weeks old, 20 in total, 10 in each group)

[0135] 2. Dosage method: The implantable osmotic pump (purchased from ALZET, USA) is an osmotic pump the size of a capsule, which meets the USP medical plastic material biocompatibility standard and can be implanted subcutaneously or intraperitoneally in experimental animals to deliver the test agent slowly, continuously and accurately at a rate of μl / hour directly or through a catheter. In this example, the pump (model: 2 weeks) was used to administer normal saline and CTRP9 229-333 polypeptide to mice.

[0136] 3. Modeling method:

[0137] refer to Figure 4 , the mice were divided into a control group and a CTRP9 229-333 group;

[0138] 2.1: Two groups of mice were fed with high-fat diet for 12 weeks to establish the diabetes model (high-fat diet was purchased from Changzhou Shusan Biotechnology Co., Ltd.).

[0139] 2.2: Myocardial ischemia-reperfusion injury treatment was performed on the two groups of mice:

[0140] 1) Male adult mice were anesthetized with 2% isoflurane;

[0141] 2) Perform a mini-left thoracotomy in the fourth intercostal space of the mouse and expose the heart;

[0142] 3) Use 6.0 silk slipknot to ligate the left coronary artery (LAD) in the middle, then quickly return the heart to the chest cavity, expel the chest gas, and quickly close the chest;

[0143] 4) After 90 minutes of ischemia, the heart was exposed again and the silk thread was untied to initiate reperfusion;

[0144] 5) The wound was then tightly sutured, the mice were cardiac reperfused for 14 days, and a high-fat diet was administered.

[0145] 2.3: An osmotic pressure drug delivery pump (purchased from Alzet) was implanted subcutaneously in two groups of mice. The drug delivery pump could deliver drugs at a rate of 0.25 μl / hour for 2 consecutive weeks. The drug delivery pump of the control group injected normal saline, and the CTRP9 229-333 group injected CTRP9229-333 polypeptide (synthesized by Gimeijiaer Biotechnology Co., Ltd. based on the sequencing results in Example 1). The drugs were delivered for two consecutive weeks to complete the establishment of the HFD+IR model.

[0146] 4. Anatomical measurement experiment

[0147] After two weeks of HFD+IR modeling and drug administration, the mice were killed and dissected, and the hearts of the mice were removed, measured and weighed. Figure 5 ,like Figure 5 As shown in the heart photo in the middle left figure, the heart volume of the control group mice was significantly larger than that of the CTRP9229-333 group mice, indicating that CTRP9 229-333 can reduce the volume of the mouse heart. Figure 5 As shown in the middle right figure, the heart-to-body weight ratio (HW / BW) of the control group mice was significantly greater than that of the CTRP9 229-333 group mice. It can be seen that the CTRP9 229-333 polypeptide can reduce the heart-to-body weight ratio of HFD+IR mice, that is, reduce the heart weight of mice.

[0148] 5. Ultrasonic experiment

[0149] 5.1 Experimental instruments:

[0150] Ultrasound High-resolution Small Animal Ultrasound 2100 imaging system (purchased from VisualSonics, Canada)

[0151] 5.2 Experimental methods:

[0152] 1) After the HFD+IR modeling was completed (i.e., two weeks after drug administration), the two groups of mice to be tested were anesthetized, and the hair on the chest of the mice was removed with a depilatory agent or a razor blade.

[0153] 2) Place the mouse with its chest hair removed on the operating table with its abdomen facing upwards, apply purified water (for conductivity) on its limbs and fix them to the corresponding positions on the operating table with tape. Adjust the operating table so that the mouse's head is slightly higher than its tail, and the apex and base of the mouse's heart are flush.

[0154] 3) Place two thick films on both sides of the mouse's chest (to fix the conductive glue), and apply the coupling agent to the mouse's chest, about 2 cm thick. The probe should be buried in the conductive glue during measurement.

[0155] 4) Detection by echocardiography system with 30-MHz imaging sensor:

[0156] First, detect the long axis of the heart. Direct the notch of the ultrasound probe toward the mouse's head, rotate it counterclockwise about 45°, and adjust the probe to fit the mouse's skin. Turn on the B-mode ultrasound mode and adjust the operation panel until the B-mode echocardiogram of the long axis of the left ventricle can be clearly observed. Then turn on the M-mode ultrasound mode, adjust the sampling line to the correct position, and press the M-mode ultrasound mode again to obtain the M-mode echocardiogram of the long axis of the left ventricle. When detecting the short axis of the heart, turn the probe notch clockwise about 90° and adjust the probe position until a clear circular echocardiogram with two papillary muscles is displayed.

[0157] 5) Data measurement and analysis:

[0158] The left ventricular ejection fraction (EF%) and the left ventricular short-axis shortening rate (FS%) were measured by echocardiography. The ejection fraction (EF%) can reflect cardiac function and remodeling, and the short-axis shortening rate (FS%) can reflect the contractility of cardiac muscles.

[0159] 5.3 Experimental Results

[0160] The cardiac ultrasound results of the two groups of HFD+IR mice were as follows Figure 6 The ejection fraction (EF%) and fractional shortening (FS%) data obtained based on ultrasound results are shown in Figure 7 Reference Figure 7Figure A shows that the left ventricular ejection fraction (EF%) of mice in the CTRP9 229-333 group was significantly higher than that in the control group; Figure 7 As shown in Figure B, the left ventricular short axis shortening rate (FS%) of the CTRP9 229-333 group of mice was significantly higher than that of the control group of mice. It can be seen that the CTRP9 229-333 peptide effectively improved the cardiac function of HFD+IR mice and enhanced their cardiac muscle contractility.

[0161] 6. Positron emission tomography (PET-CT) experiment

[0162] Small animal PET / CT performs functional and anatomical imaging of rodents (mice or rats) in vivo to obtain various data on the animal's physical condition and the distribution of drugs in the small animal. The heart is a complex, constantly beating organ, and obtaining clear cardiac images requires high spatial and temporal resolution. PET is the gold standard for non-invasive assessment of myocardial activity and can assess coronary artery disease through myocardial perfusion; at the same time, 18 F-FDG is a tracer with specific biological activity. 18 F is a radioactive nuclide, and FDG is a glucose analogue, which is used to make organs in the body produce glucose metabolism. It can reflect cardiovascular physiological and biochemical information from different angles, and has high sensitivity and repeatability. It has been used for myocardial perfusion imaging to evaluate cardiac blood flow distribution, diagnose myocardial ischemia and myocardial infarction, determine the location and range of lesions, and observe ventricular wall motion. In addition, myocardial imaging can be used to understand the local blood perfusion and metabolism of the heart.

[0163] 6.1 Experimental methods:

[0164] 1) After the HFD+IR modeling was completed (i.e., two weeks after administration), the two groups of mice were fasted for 6 hours, anesthetized with 2% isoflurane, and injected through the tail vein of the mice 18 F-FDG.

[0165] 2) During injection 18 After 20 minutes of F-FDG, the small animal PET / CT system (purchased from Siemens, USA) was used to acquire images to capture 18 The distribution of F-FDG in the body.

[0166] 3) The small animal PET / CT system is capable of acquiring 60 minutes of ECG-gated list-mode PET dynamic data, allowing for accurate assessment of temporal changes in tracer uptake in the myocardium.

[0167] 4) A CT scan was then performed, which obtained 120 projections through continuous rotation, covering an angular range of 220°. When performing the CT scan, the imaging parameters were carefully set, using an 80 kilovolt peak (kVp) X-ray tube, 0.5 milliamperes (mA) current, and 160 milliseconds (ms) exposure time to ensure high-quality anatomical images.

[0168] 5) PET and CT images were automatically fused and analyzed using the manufacturer's software (Inveon Acquisition Workplace 1.4, Siemens Medical Systems, USA).

[0169] 6.2 Experimental Results

[0170] The PET-CT results of the two groups of HFD+IR mice are as follows Figure 8 The standard uptake value (SUV) data calculated based on PET / CT results are shown in Fig. 9 Reference Figure 8 , the red area in the figure is healthy heart tissue. The PET-CT test results of normal mouse heart are approximately circular red areas, which means normal heart function and normal glucose metabolism. When the mouse heart is ischemic or infarcted, the infarcted or ischemic area cannot metabolize glucose normally, and the infarcted or ischemic area detected by PET-CT cannot be displayed, and the whole cannot be displayed as a nearly circular red area. The more complete the heart area shown by PET-CT test, the more normal the heart is and the better the function. The cross-sectional area of ​​healthy heart tissue in mice in the CTRP9229-333 group was significantly larger than that in mice in the control group, indicating that the polypeptide significantly reduced the area of ​​myocardial infarction. Reference Fig. 9 The SUV value of the CTRP9 229-333 group was significantly higher than that of the control group, indicating that the cross-sectional area of ​​healthy cardiac tissue was large and the active areas were more, as well as the metabolic activity was enhanced.

[0171] In summary, CTRP9 229-333 peptide has a significant protective effect on cardiac function in HFD+IR model mice by significantly reducing the myocardial infarction area, further emphasizing its potential therapeutic value in cardiac pathological states. CTRP9229-333 peptide significantly improved the size of myocardial infarction area in HFD+IR mice.

[0172] 7. Vevo strain analysis experiment

[0173] Vevo strain analysis is a technique that uses ultrasound biomicroscopy (UBM) technology to analyze images through Vevo strain software to quantitatively evaluate myocardial wall strain. This technique can non-invasively and easily evaluate mechanical parameters such as myocardial strain, strain rate, and strain peak time, and is suitable for cardiac function research in small animals such as mice.

[0174] 7.1 Experimental methods:

[0175] 1) After the HFD+IR modeling was completed (i.e., two weeks after drug administration), the two groups of mice to be tested were anesthetized, and the hair on the chest of the mice was removed with a depilatory agent or a razor blade.

[0176] 2) Place the mouse with its chest hair removed on the operating table with its abdomen facing upwards, apply purified water (for conductivity) on its limbs and fix them to the corresponding positions on the operating table with tape. Adjust the operating table so that the mouse's head is slightly higher than its tail, and the apex and base of the mouse's heart are flush.

[0177] 3) Place two thick films on both sides of the mouse's chest (to fix the conductive glue), and apply the coupling agent to the mouse's chest, about 2 cm thick. The probe should be buried in the conductive glue during measurement.

[0178] 4) Detection by echocardiography system with 30-MHz imaging sensor: Detect the long axis of the heart, turn the ultrasound probe notch toward the mouse head, rotate counterclockwise about 45°, and adjust the probe to fit the mouse skin. Turn on the B-mode ultrasound mode and adjust the operation panel until the B-mode echocardiogram of the long axis of the left ventricle can be clearly observed.

[0179] 5) Image processing and analysis: The speckle tracking technology in Vevo Strain software is used to identify tiny spots in myocardial tissue and track the movement trajectory of these spots during the cardiac cycle to evaluate the deformation and contractility of the myocardium. Based on the results of speckle tracking, the strain parameters of the myocardium are calculated, including radial strain (Radial Strain and Radial Strain Rate) and longitudinal strain (Global Longitudinal Strain, Longitudinal Strain, and Longitudinal Strain Rate). Radial strain reflects the degree of deformation of the myocardium in the radial direction, while longitudinal strain reflects the degree of deformation of the myocardium in the longitudinal direction.

[0180] 7.2 Experimental results:

[0181] The results of cardiac Vevo strain analysis of the two groups of HFD+IR mice are shown in Figure 2. Fig.10 The experimental data of Vevo strain analysis are shown in Fig.11 Refer to Fig.11Figures A, D, and E in the figure show that the global longitudinal strain, longitudinal strain, and longitudinal strain rate of the CTRP9 229-333 group of mice were significantly lower than those of the control group, indicating that the longitudinal myocardial deformability of this group of mice was weaker; Fig.11 As shown in Figures B and C, the radial strain (Radial Strain) and radial strain rate (Radial Strain Rate) of the hearts of mice in the CTRP9 229-333 group were significantly higher than those in the control group, indicating that their radial myocardial function was significantly improved.

[0182] These results indicate that CTRP9 229-333 peptide can effectively improve the cardiac diastolic function of HFD+IR model mice, especially in terms of enhancing radial myocardial strain capacity, further confirming the potential therapeutic role of this peptide in regulating cardiac mechanical function and responding to cardiac pathological damage. It can be seen that CTRP9 229-333 peptide effectively improved the cardiac diastolic function of HFD+IR mice.

[0183] 8. Masson staining experiment

[0184] Masson staining, also known as Masson staining, is a classic and authoritative technical method in the field of connective tissue staining, focusing on the staining of collagen fibers. This method is widely used in histological research. Its main purpose is to stain the nucleus and selectively display collagen fibers and muscle fibers. The principle of Masson staining is closely related to the size of anionic dye molecules and their permeability to tissues: dyes with small molecular weight can easily penetrate densely structured and low permeability tissues, while large molecular weight dyes can only enter loosely structured and highly permeable tissues. Due to the large molecular weight of aniline blue, after Masson staining, muscle fibers appear red, while collagen fibers appear blue.

[0185] In this experiment, Masson staining was performed on the heart sections of HFD+IR model mice. Normal myocardial tissue will appear red after staining, and fibrotic tissue will appear blue after staining. The effect of CTRP9 229-333 peptide on cardiac fibrosis was verified by analyzing the proportion of fibrotic tissue area.

[0186] 8.1 Experimental Materials

[0187] 4% paraformaldehyde, cycad hematoxylin staining solution, Ponceau red-acid fuchsin staining solution, phosphomolybdic acid and aniline blue (all purchased from Beijing Solebow Technology Co., Ltd.), xylene and anhydrous ethanol (all purchased from Beijing Dongfang Boyuan Technology Development Co., Ltd.).

[0188] 8.2 Experimental Methods

[0189] 1) Fixation, dehydration, embedding and sectioning: After modeling, HFD+IR mice were killed and dissected, the hearts of the mice were removed, washed with PBS, and fixed in 4% paraformaldehyde in a 4°C refrigerator for 48 hours. The heart tissues were routinely dehydrated and embedded, and then sectioned after being fixed in paraffin.

[0190] 2) Dewaxing of paraffin sections: soak the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally wash with distilled water.

[0191] 3) Cycad hematoxylin staining: Immerse the sections in cycad hematoxylin staining solution for 3-8 minutes, then rinse with running water.

[0192] 4) Ponceau-acid fuchsin staining: The sections were placed in a Ponceau-acid fuchsin staining solution for 5 minutes and then rinsed with 0.2% glacial acetic acid.

[0193] 5) Phosphomolybdic acid differentiation: Place the slices in phosphomolybdic acid for differentiation for a few seconds and then wash with water.

[0194] 6) Aniline blue staining: Place the sections in aniline blue staining solution for 5 minutes and then rinse with 0.2% glacial acetic acid.

[0195] 7) Rapid dehydration and transparentization: Soak the slices in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes. Then take the slices out of xylene and let them dry slightly. Finally, use neutral gum to seal the slices.

[0196] 8) Microscopic examination, image acquisition and analysis.

[0197] 8.3 Experimental Results

[0198] The results of the Masson staining experiment of the heart tissue of the two groups of HFD+IR mice are shown in Fig.12 As shown, reference Fig.12 Left: At the same microscopic scale, the blue area of ​​the heart tissue of the CTRP9 229-333 group of mice was significantly smaller than that of the control group of mice. As mentioned above, the blue area is the fibrotic tissue; Fig.12In the right figure, the area of ​​fibrosis in the heart tissue of mice in the CTRP9 229-333 group was significantly lower than that in the control group. It can be seen that the CTRP9 229-333 peptide can effectively inhibit cardiac fibrosis in HFD+IR mice.

[0199] 9. WGA staining experiment

[0200] Wheat germ agglutinin (WGA, Triticum vulgaris lectin) is extracted from grains and can specifically bind to a glycoprotein on the myocardial cell membrane. WGA can stain the myocardial cell membrane, so as to visually see whether the myocardial cell is hypertrophic or not, and to facilitate the use of software to measure the diameter and area of ​​the stained myocardial cells. In this experiment, WGA staining was performed on the heart slices of HFD+IR model mice, and the effect of CTRP9 229-333 peptide on myocardial hypertrophy was verified by analyzing the area of ​​myocardial cells.

[0201] 9.1 Experimental Materials

[0202] 4% paraformaldehyde (purchased from Beijing Solebow Technology Co., Ltd.), xylene and anhydrous ethanol (purchased from Beijing Dongfang Boyuan Technology Development Co., Ltd.), citrate antigen retrieval solution and WGA staining solution (purchased from Wuhan Sevier Biotechnology Co., Ltd.), and DAPI (purchased from Thermo Fisher Scientific, USA).

[0203] 9.2 Experimental Methods

[0204] 1) Fixation, dehydration, embedding and sectioning: After modeling, HFD+IR mice were killed and dissected, the hearts of the mice were removed, washed with PBS, and fixed in 4% paraformaldehyde in a 4°C refrigerator for 48 hours. The heart tissues were routinely dehydrated and embedded, and then sectioned after being fixed in paraffin.

[0205] 2) Dewaxing of paraffin sections: soak the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally wash with distilled water.

[0206] 3) Antigen repair: The tissue sections were placed in citrate antigen repair solution (pH 6.0) and antigen repair was performed in a pressure cooker. After boiling for 5 minutes, the sections were taken out, cooled naturally to room temperature, and washed twice with distilled water.

[0207] 4) WGA staining: Add 50-100 μl of diluted WGA working solution to each section, incubate at room temperature in the dark for 30 minutes, and rinse three times with PBS after incubation.

[0208] 5) DAPI nuclear staining: add 50-100 μl of diluted DAPI working solution to each section, incubate at room temperature in the dark for 5 minutes, and rinse three times with PBS after incubation.

[0209] 6) Sealing: Seal the slides with anti-fluorescence quenching sealing medium and store at 4°C away from light.

[0210] 7) Microscopic examination, image acquisition and analysis.

[0211] 9.3 Experimental Results

[0212] The results of WGA staining experiments on the heart tissues of the two groups of HFD+IR mice are shown in Figure 2. Fig.13 As shown in the figure, at a microscopic scale of 50 μm, the cross-sectional area of ​​cardiomyocytes in the CTRP9 229-333 group of mice was significantly smaller than that in the control group of mice. It can be seen that the CTRP9 229-333 polypeptide can effectively inhibit the hypertrophy of cardiomyocytes in HFD+IR mice.

[0213] 10. Reverse transcription real-time quantitative PCR (qRT-PCR) experiment

[0214] CTGF (connective tissue growth factor) promotes fibrosis by enhancing fibroblast activation and ECM protein synthesis; Col3α1 (type III collagen α1 chain) encodes type III collagen, which is the main component of ECM. Overexpression leads to collagen deposition and tissue stiffness; TGFβ (transforming growth factor-β) is the main regulator of fibrosis, regulating the generation of ECM and the expression of fibrotic factors through the Smad signaling pathway. The three interact with each other to jointly regulate ECM accumulation, extracellular matrix remodeling, and changes in tissue structure during the pathological process of fibrosis, and are therefore considered to be key regulatory genes for tissue fibrosis.

[0215] ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide) are markers of cardiac pressure load response, and their up-regulated expression is a compensatory mechanism for myocardial hypertrophy and cardiac remodeling; ACTA1 (α-actinin 1) is a key component of myocardial cell structure, supporting the myocardial response to mechanical stress during hypertrophy; Rcan1.4 (calcineurin inhibitor 1.4) inhibits excessive hypertrophic response of myocardial cells by regulating the calcineurin signaling pathway; the up-regulated expression of β-MHC (β-myosin heavy chain) reflects the decline of myocardial contractile function during pathological myocardial remodeling. These genes participate in and mark the occurrence and progression of myocardial hypertrophy by regulating the metabolism, structure and signal transduction of myocardial cells, and are therefore widely used as molecular markers in the study of myocardial hypertrophy.

[0216] 10.1 Experimental Reagents

[0217] RNA extraction kit (purchased from Thermo Fisher Scientific, USA), reverse transcription reaction solution and PCR reaction solution (both purchased from Baoriyi Biotechnology Co., Ltd., Japan).

[0218] 10.2 Experimental Methods

[0219] Total RNA was extracted using an RNA extraction kit, and a reverse transcription reaction solution was prepared to reverse transcribe the extracted RNA into cDNA. A PCR reaction solution was prepared according to the reaction system. PCR amplification was then performed on a 7500 Real-Time PCR Syetem (purchased from Thermo Fisher Scientific, USA) according to the following conditions:

[0220] 1) 95°C for 10 seconds, 1 cycle;

[0221] 2) 95°C for 5 seconds, 60°C for 20 seconds, 40 cycles;

[0222] 3) 95℃ for 1 minute, 55℃ for 30 seconds, 95℃ for 30 seconds, 1 cycle

[0223] After the reaction is completed, each reaction tube can obtain a CT value, using 2 -ΔΔCt The method calculates the result according to the following formula:

[0224] ΔΔCt=ΔCt(targetgene)-ΔCt(control);

[0225] ΔCt(target gene)=Ct(experimental target gene)-Ct(control target gene);

[0226] ΔCt(control)=Ct(experimental control)-Ct(control).

[0227] Statistical analysis was performed by Graph prism 8.0 (GraphPad Software, San Diego, CA).

[0228] 10.2 Experimental Results

[0229] The experimental data of the mRNA levels of CTGF, Col3α1, and TGFβ in the heart tissues of the two groups of HFD+IR mice were detected by qRT-PCR. Fig.14 As shown, refer to Fig.14As shown in Figures A, B, and C, the mRNA levels of CTGF, Col3α1, and TGFβ in the heart tissues of mice in the CTRP9229-333 group were significantly lower than those in the control group. It can be seen that the CTRP9229-333 polypeptide effectively inhibited the expression of cardiac fibrosis genes, thereby inhibiting cardiac fibrosis in HFD+IR mice.

[0230] The experimental data of the mRNA levels of five genes, ANP, BNP, ACTA1, Rcan1.4, and β-MHC, detected by qRT-PCR in the heart tissues of the two groups of HFD+IR mice are as follows Fig.15 As shown, refer to Fig.15 From Figures A to E in Figure 1, the mRNA levels of five genes, ANP, BNP, ACTA1, Rcan1.4, and β-MHC, in the heart tissues of mice in the CTRP9 229-333 group were significantly lower than those in the control group. It can be seen that the CTRP9 229-333 polypeptide effectively inhibited the expression of cardiac hypertrophy genes, thereby inhibiting the hypertrophy of cardiomyocytes in HFD+IR mice.

[0231] In summary, CTRP9 229-333 peptide can reduce the heart weight, improve cardiac function, improve the size of myocardial infarction area, and improve cardiac diastolic function of HFD+IR mice. It can be seen that CTRP9 229-333 peptide has an inhibitory effect on myocardial remodeling caused by ischemia-reperfusion in HFD+IR mice.

[0232] In addition, CTRP9 229-333 peptide can inhibit cardiac fibrosis in HFD+IR mice by inhibiting the expression of cardiac fibrosis genes, and inhibit cardiomyocyte hypertrophy in HFD+IR mice by inhibiting the expression of cardiac hypertrophy genes. Since the main pathological mechanisms of myocardial remodeling include myocardial fibrosis caused by fibroblast proliferation and extracellular matrix (ECM) reorganization, and myocardial hypertrophy caused by reduced cardiac contractility through mitochondrial dysfunction and metabolic abnormalities, and CTRP9 229-333 peptide can inhibit the expression of cardiac fibrosis genes and hypertrophy genes, it further shows that CTRP9 229-333 peptide can inhibit myocardial remodeling.

[0233] Example 4: Effect of CTRP9 229-333 polypeptide in heart failure model with preserved ejection fraction (HFpEF)

[0234] Heart failure (HF) refers to a group of complex clinical syndromes caused by cardiac structural or functional diseases that result in cardiac output failing to meet the body's needs, with the main manifestations being dyspnea, excessive fatigue, and lower limb edema. Currently, it can be divided into three categories based on left ventricular ejection fraction (LVEF), namely heart failure with reduced ejection fraction (HFrEF), heart failure with mildly reduced ejection fraction (HFmrEF), and heart failure with preserved ejection fraction (HFpEF), among which HFrEF and HFpEF account for the highest proportion.

[0235] Heart failure with preserved ejection fraction (HFpEF) is caused by metabolic changes caused by aging, hypertension, obesity and diabetes, as well as widespread chronic inflammation. In HFpEF, the endothelial cells that line the small blood vessels of the heart are dysfunctional and cannot synthesize sufficient amounts of nitric oxide, a substance involved in vasodilation. Due to changes in proteins involved in molecular signaling pathways and cell contraction, the cardiomyocytes themselves become more rigid. The end result is slow filling of the heart. In this example, myocardial damage was caused in mice by establishing heart failure with preserved ejection fraction (HFpEF), thereby verifying the protective effect of CTRP9 229-333 polypeptide on myocardial damage and the therapeutic effect on myocardial remodeling.

[0236] 1. Experimental animals: Wild-type mice (purchased from Beijing Huafukang Experimental Animal Company, strain C57BL / 6J, male, 8 weeks old, 20 in total, 10 in each group)

[0237] 2. Modeling method:

[0238] refer to Fig.16 , the mice were divided into a control group and a CTRP9 229-333 group;

[0239] 2.1: Two groups of mice were fed with high-fat diet (high-fat diet purchased from Changzhou Shusan Biotechnology Co., Ltd.) for 7 weeks. The high-fat diet made the mice obese and glucose-intolerant.

[0240] 2.2: Two groups of mice were fed a high-fat diet + L-NAME (purchased from Merck Biotech, Germany) for 3 weeks. L-NAME is Nω-nitro-L-arginine methyl ester. This drug can inhibit an enzyme that is normally expressed at a constant level (constive-no synthase), thereby catalyzing the production of nitric oxide, thereby inducing hypertension in mice and causing the mice to suffer from HFpEF.

[0241] 2.3: An osmotic pressure drug delivery pump (purchased from Alzet) was implanted subcutaneously in two groups of mice. The drug delivery pump could deliver drugs continuously at a rate of 0.25 μl / hour for 2 consecutive weeks. The drug delivery pump of the control group injected normal saline, and the CTRP9 229-333 group injected CTRP9229-333 polypeptide (synthesized by Gilead Biotechnology Co., Ltd. based on the sequencing results in Example 1). The drugs were delivered continuously for two weeks to complete the establishment of the HFpEF model.

[0242] 3. Anatomical measurement experiment

[0243] After HFpEF modeling was completed (i.e., two weeks after drug administration), the mice were killed and dissected, and the hearts, lungs, and tibias of the mice were removed, measured, and weighed. Fig.17 ,like Fig.17 As shown in the heart photograph in Figure A, the heart volume of the control group mice was significantly larger than that of the CTRP9 229-333 group mice, indicating that CTRP9 229-333 can reduce the volume of the mouse heart.

[0244] like Fig.17 As shown in Figure B, the heart-to-body weight ratio (HW / BW) of the control group mice was significantly greater than that of the CTRP9 229-333 group mice, indicating that the CTRP9 229-333 polypeptide can reduce the heart-to-body weight ratio of HFpEF mice; Fig.17 As shown in Figure D, the heart weight / tibia length of the control group mice was significantly greater than that of the CTRP9 229-333 group mice, indicating that the CTRP9 229-333 polypeptide can reduce the heart weight / tibia length of HFpEF mice; it can be seen that the CTRP9 229-333 polypeptide can reduce the heart weight of mice.

[0245] like Fig.17 As shown in Figure C, the lung weight to body weight ratio (LW / BW) of the control group mice was greater than that of the CTRP9 229-333 group mice. It can be seen that the CTRP9 229-333 polypeptide can reduce the lung weight to body weight ratio of HFpEF mice, that is, reduce the lung weight of mice.

[0246] 4. In vivo measurement experiment

[0247] 4.1 Experimental Methods

[0248] 4.1.1 Exercise capacity measurement experiment: One of the typical symptoms of HFpEF patients is exercise intolerance, which is manifested as fatigue and dyspnea during daily activities. By measuring the exercise performance of mice on a treadmill, we can simulate human exercise endurance tests and evaluate whether the HFpEF model successfully simulates these symptoms. This part of the experiment was completed using a small animal treadmill (purchased from Beijing Jitaiyuancheng Technology Co., Ltd.)

[0249] 1) Treadmill training of mice: Both groups of mice ran on a treadmill for 3 days, 60 minutes a day. The treadmill inclination angle was maintained at 10 degrees, and the speed started at 10 m / min, then increased by 1 m / min every 5 minutes until it reached 20 m / min and maintained until the end of the training.

[0250] 2) Exercise capacity measurement: After three days of adaptation to treadmill exercise, the test officially began. The treadmill started at 10 m / min and the inclination angle started at 0°. After that, the inclination angle increased by 10° every 5 minutes until it reached a maximum of 30°; at the same time, the treadmill speed increased by 1 m / min every 3 minutes until all mice were exhausted. The exhaustion standard was defined as the inability of the mouse to resume running within 10 seconds after direct contact with the electric shock net. The running time was measured during the test and the running distance was recorded.

[0251] 3) Compare the running distance of each group of mice to evaluate the mice's athletic ability.

[0252] 4.1.2 Blood glucose measurement: The mice were fasted for 5 hours before measurement. Then, the tail of the mice was cut off by scissors, about 2 mm, and the first drop of blood was discarded. The blood was then dripped onto a blood glucose test strip, and the collected blood was measured using a blood glucose meter, and the blood glucose value was recorded.

[0253] 4.2 Experimental Results

[0254] The results of the exercise capacity and blood glucose test of the two groups of HFpEF mice are as follows Fig.18 As shown, reference Fig.18 As shown in Figure A, the exercise capacity of mice in the CTRP9 229-333 group was stronger than that of mice in the control group. This shows that the CTRP9 229-333 peptide can improve the exercise capacity of HFpEF mice.

[0255] refer to Fig.18 As shown in Figure B, there was no significant difference in blood glucose between the CTRP9 229-333 group and the control group, and both groups maintained a high blood glucose level. This shows that HFpEF modeling was successful and the CTRP9 229-333 polypeptide did not affect the blood glucose of HFpEF mice.

[0256] 5. Ultrasonic experiment

[0257] 5.1. Experimental instruments: Ultrasound high-resolution small animal ultrasound 2100 imaging system (purchased from VisualSonics, Canada).

[0258] 5.2 Experimental methods:

[0259] 1) After HFpEF modeling was completed (i.e., two weeks after drug administration), the two groups of mice to be tested were anesthetized, and the hair on the chest of the mice was removed with a depilatory agent or a razor blade.

[0260] 2) Place the mouse with its chest hair removed on the operating table with its abdomen facing upwards, apply purified water (for conductivity) on its limbs and fix them to the corresponding positions on the operating table with tape. Adjust the operating table so that the mouse's head is slightly higher than its tail, and the apex and base of the mouse's heart are flush.

[0261] 3) Place two thick films on both sides of the mouse's chest (to fix the conductive glue), and apply the coupling agent to the mouse's chest, about 2 cm thick. The probe should be buried in the conductive glue during measurement.

[0262] 4) Detection by an echocardiography system with a 30-MHz imaging sensor: First, detect the long axis of the heart, point the ultrasound probe notch toward the mouse head, rotate it counterclockwise about 45°, and adjust the probe to fit the mouse skin. Turn on the B-mode ultrasound mode and adjust the operation panel until the B-mode echocardiogram of the long axis of the left ventricle can be clearly observed. Then turn on the M-mode ultrasound mode, adjust the sampling line to the correct position, and press the M-mode ultrasound mode again to obtain the M-mode echocardiogram of the long axis of the left ventricle. When detecting the short axis of the heart, turn the probe notch clockwise about 90° and adjust the probe position until a clear circular cardiogram with two papillary muscles is displayed. Then the ultrasound probe cuts from the apex toward the mouse head and adjusts the operation panel until the four heart chambers of the mouse can be clearly observed. Then, pulsed Doppler or tissue Doppler technology was used to measure the early diastolic peak blood flow velocity (E), late diastolic peak blood flow velocity (A), and early diastolic blood flow velocity (E') and late peak blood flow velocity (A') of the mitral annulus, and parameters such as E / A, E / E' ratio and isovolumetric relaxation time (IVRT) were calculated to evaluate cardiac diastolic function.

[0263] 5) Data measurement and analysis: E / A, E / E' and isovolumetric relaxation time (IVRT) are calculated by tissue Doppler imaging (TDI) spectrum measurement. Among them, E / A mainly reflects the diastolic function of the heart, specifically the ratio of the E peak to the A peak of the mitral valve forward blood flow. The E peak is the first peak of blood flow from the left atrium to the left ventricle. Due to the decrease in pressure during left ventricular diastole, the blood from the left atrium is sucked into the left ventricle, representing the rapid diastole, i.e., the early diastole; the A peak is the second peak of blood flow from the left atrium to the left ventricle. It is the contraction of the atrium itself, which pushes the blood from the left atrium into the left ventricle, representing the atrial contraction, i.e., the late diastole. E / E' is generally used to evaluate the left ventricular diastolic function and the estimation of left ventricular filling pressure, where E represents the peak flow velocity in the early diastole of the heart, and E' represents the peak velocity wave in the early diastole of the heart. The isovolumetric relaxation time (IVRT) is mainly used to measure the diastolic function of the aortic valve, mitral valve, etc.

[0264] 5.3 Experimental Results

[0265] Since the main pathological feature of HFpEF is cardiac diastolic dysfunction, ultrasound showed that the cardiac systolic function of HFpEF mice was normal, but the diastolic function was altered. Therefore, this experiment focused on the effect of CTRP9 229-333 peptide on the diastolic function of HFpEF model mice. Fig.19 As shown, the E / A, E / E' and IVRT data obtained based on ultrasound results are as follows Fig. 20 Reference Fig. 20 Figure A shows that the E / A of CTRP9 229-333 mice was significantly higher than that of the control mice; Figure 7 Figure B shows that the E / E' of CTRP9 229-333 mice was lower than that of the control mice; Figure 7 As shown in Figure C, the IVRT of mice in the CTRP9 229-333 group was lower than that of mice in the control group. It can be seen that the CTRP9 229-333 peptide effectively improved the cardiac diastolic function of HFpEF mice.

[0266] 6. Vevo strain analysis experiment

[0267] Vevo strain analysis is a technique that uses ultrasound biomicroscopy (UBM) technology to analyze images through Vevo strain software to quantitatively evaluate myocardial wall strain. This technique can non-invasively and easily evaluate mechanical parameters such as myocardial strain, strain rate, and strain peak time, and is suitable for cardiac function research in small animals such as mice.

[0268] 6.1 Experimental methods:

[0269] 1) After the HFD+IR modeling was completed (i.e., two weeks after drug administration), the two groups of mice to be tested were anesthetized, and the hair on the chest of the mice was removed with a depilatory agent or a razor blade.

[0270] 2) Place the mouse with its chest hair removed on the operating table with its abdomen facing upwards, apply purified water (for conductivity) on its limbs and fix them to the corresponding positions on the operating table with tape. Adjust the operating table so that the mouse's head is slightly higher than its tail, and the apex and base of the mouse's heart are flush.

[0271] 3) Place two thick films on both sides of the mouse's chest (to fix the conductive glue), and apply the coupling agent to the mouse's chest, about 2 cm thick. The probe should be buried in the conductive glue during measurement.

[0272] 4) Detection by echocardiography system with 30-MHz imaging sensor: Detect the long axis of the heart, turn the ultrasound probe notch toward the mouse head, rotate counterclockwise about 45°, and adjust the probe to fit the mouse skin. Turn on the B-mode ultrasound mode and adjust the operation panel until the B-mode echocardiogram of the long axis of the left ventricle can be clearly observed.

[0273] 5) Image processing and analysis: The speckle tracking technology in Vevo Strain software is used to identify tiny spots in myocardial tissue and track the movement trajectory of these spots during the cardiac cycle to evaluate the deformation and contractility of the myocardium. Based on the results of speckle tracking, the strain parameters of the myocardium are calculated, including radial strain (Radial Strain and Radial Strain Rate) and longitudinal strain (Global Longitudinal Strain, Longitudinal Strain, and Longitudinal Strain Rate). Radial strain reflects the degree of deformation of the myocardium in the radial direction, while longitudinal strain reflects the degree of deformation of the myocardium in the longitudinal direction.

[0274] 6.2 Experimental results:

[0275] The results of cardiac Vevo strain analysis of the two groups of HFpEF mice are shown in Fig.21 The experimental data of Vevo strain analysis are shown in Fig. 22 Reference Fig. 22 The global longitudinal strain of the heart of mice in the CTRP9 229-333 group was significantly lower than that in the control group. It can be seen that the CTRP9 229-333 peptide effectively improved the cardiac diastolic function of HFpEF mice.

[0276] 7. Masson staining experiment

[0277] 7.1 Experimental Materials

[0278] 4% paraformaldehyde, cycad hematoxylin staining solution, Ponceau red-acid fuchsin staining solution, phosphomolybdic acid and aniline blue (all purchased from Beijing Solebow Technology Co., Ltd.), xylene and anhydrous ethanol (all purchased from Beijing Dongfang Boyuan Technology Development Co., Ltd.).

[0279] 7.2 Experimental Methods

[0280] 1) Fixation, dehydration, embedding and sectioning: After modeling, HFpEF mice were killed and dissected, the hearts of the mice were removed, washed with PBS, and fixed in 4% paraformaldehyde in a 4°C refrigerator for 48 hours. The heart tissues were routinely dehydrated and embedded, and then sectioned after being fixed in paraffin.

[0281] 2) Dewaxing of paraffin sections: soak the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally wash with distilled water.

[0282] 3) Cycad hematoxylin staining: Immerse the sections in cycad hematoxylin staining solution for 3-8 minutes, then rinse with running water.

[0283] 4) Ponceau-acid fuchsin staining: The sections were placed in a Ponceau-acid fuchsin staining solution for 5 minutes and then rinsed with 0.2% glacial acetic acid.

[0284] 5) Phosphomolybdic acid differentiation: Place the slices in phosphomolybdic acid for differentiation for a few seconds and then wash with water.

[0285] 6) Aniline blue staining: Place the sections in aniline blue staining solution for 5 minutes and then rinse with 0.2% glacial acetic acid.

[0286] 7) Rapid dehydration and transparentization: Soak the slices in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes. Then take the slices out of xylene and let them dry slightly. Finally, use neutral gum to seal the slices.

[0287] 8) Microscopic examination, image acquisition and analysis.

[0288] 7.3 Experimental Results

[0289] The results of the Masson staining experiment of the heart tissue of the two groups of HFpEF mice are shown in Fig.23 As shown, reference Fig.23Left: At the same microscopic scale, the blue area of ​​the heart tissue of the CTRP9 229-333 group of mice was significantly smaller than that of the control group of mice. As mentioned above, the blue area is the fibrotic tissue; Fig.23 In the right figure, the area of ​​fibrosis in the heart tissue of mice in the CTRP9 229-333 group was significantly lower than that in the control group. It can be seen that the CTRP9 229-333 peptide can effectively inhibit cardiac fibrosis in HFpEF mice.

[0290] 8. WGA staining experiment

[0291] 8.1 Experimental Materials

[0292] 4% paraformaldehyde (purchased from Beijing Solebow Technology Co., Ltd.), xylene and anhydrous ethanol (purchased from Beijing Dongfang Boyuan Technology Development Co., Ltd.), citrate antigen retrieval solution and WGA staining solution (purchased from Wuhan Sevier Biotechnology Co., Ltd.), and DAPI (purchased from Thermo Fisher Scientific, USA).

[0293] 8.2 Experimental Methods

[0294] 1) Fixation, dehydration, embedding and sectioning: After modeling, HFpEF mice were killed and dissected, the hearts of the mice were removed, washed with PBS, and fixed in 4% paraformaldehyde in a 4°C refrigerator for 48 hours. The heart tissues were routinely dehydrated and embedded, and then sectioned after being fixed in paraffin.

[0295] 2) Dewaxing of paraffin sections: soak the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally wash with distilled water.

[0296] 3) Antigen repair: The tissue sections were placed in citrate antigen repair solution (pH 6.0) and antigen repair was performed in a pressure cooker. After boiling for 5 minutes, the sections were taken out, cooled naturally to room temperature, and washed twice with distilled water.

[0297] 4) WGA staining: Add 50-100 μl of diluted WGA working solution to each section, incubate at room temperature in the dark for 30 minutes, and rinse three times with PBS after incubation.

[0298] 5) DAPI nuclear staining: add 50-100 μl of diluted DAPI working solution to each section, incubate at room temperature in the dark for 5 minutes, and rinse three times with PBS after incubation.

[0299] 6) Sealing: Seal the slides with anti-fluorescence quenching sealing medium and store at 4°C away from light.

[0300] 7) Microscopic examination, image acquisition and analysis.

[0301] 8.3 Experimental Results

[0302] The results of WGA staining experiments on cardiac tissues of the two groups of HFpEF mice are shown in Figure 2. Fig.24 As shown in the figure, at a microscopic scale of 50 μm, the cross-sectional area of ​​cardiomyocytes in the CTRP9 229-333 group of mice was significantly smaller than that in the control group of mice. It can be seen that the CTRP9 229-333 polypeptide can effectively inhibit the hypertrophy of cardiomyocytes in HFpEF mice.

[0303] 9. Reverse transcription real-time quantitative PCR (qRT-PCR) experiment

[0304] CTGF (connective tissue growth factor) promotes fibrosis by enhancing fibroblast activation and ECM protein synthesis; Col3α1 (type III collagen α1 chain) encodes type III collagen, which is the main component of ECM. Overexpression leads to collagen deposition and tissue stiffness; TGFβ (transforming growth factor-β) is the main regulator of fibrosis, regulating the generation of ECM and the expression of fibrotic factors through the Smad signaling pathway. The three interact with each other to jointly regulate ECM accumulation, extracellular matrix remodeling, and changes in tissue structure during the pathological process of fibrosis, and are therefore considered to be key regulatory genes for tissue fibrosis.

[0305] ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide) are markers of cardiac pressure load response, and their up-regulated expression is a compensatory mechanism for myocardial hypertrophy and cardiac remodeling; ACTA1 (α-actinin 1) is a key component of myocardial cell structure, supporting the myocardial response to mechanical stress during hypertrophy; Rcan1.4 (calcineurin inhibitor 1.4) inhibits excessive hypertrophic response of myocardial cells by regulating the calcineurin signaling pathway; the up-regulated expression of β-MHC (β-myosin heavy chain) reflects the decline of myocardial contractile function during pathological myocardial remodeling. These genes participate in and mark the occurrence and progression of myocardial hypertrophy by regulating the metabolism, structure and signal transduction of myocardial cells, and are therefore widely used as molecular markers in the study of myocardial hypertrophy.

[0306] 9.1 Experimental Methods

[0307] Total RNA was extracted using an RNA extraction kit, and a reverse transcription reaction solution was prepared to reverse transcribe the extracted RNA into cDNA. A PCR reaction solution was prepared according to the reaction system. PCR amplification was then performed on a 7500 Real-Time PCR Syetem (purchased from Thermo Fisher Scientific, USA) according to the following conditions:

[0308] 1) 95°C for 10 seconds, 1 cycle;

[0309] 2) 95°C for 5 seconds, 60°C for 20 seconds, 40 cycles;

[0310] 3) 95°C for 1 min, 55°C for 30 sec, 95°C for 30 sec, 1 cycle.

[0311] After the reaction is completed, each reaction tube can obtain a CT value, using 2 -ΔΔCt The method calculates the result according to the following formula:

[0312] ΔΔCt=ΔCt(targetgene)-ΔCt(control);

[0313] ΔCt(target gene)=Ct(experimental target gene)-Ct(control target gene);

[0314] ΔCt(control)=Ct(experimental control)-Ct(control).

[0315] Statistical analysis was performed by Graph prism 8.0 (GraphPad Software, San Diego, CA).

[0316] 9.2 Experimental Results

[0317] The experimental data of the mRNA levels of CTGF, Col3α1, and TGFβ genes detected by qRT-PCR in the heart tissues of the two groups of HFpEF mice are as follows Fig.25 As shown, refer to Fig.25 As shown in Figures A, B, and C, the mRNA levels of CTGF, Col3α1, and TGFβ in the heart tissues of mice in the CTRP9229-333 group were significantly lower than those in the control group. It can be seen that the CTRP9229-333 polypeptide effectively inhibited the expression of cardiac fibrosis genes, thereby inhibiting cardiac fibrosis in HFpEF mice.

[0318] The experimental data of the mRNA levels of five genes, ANP, BNP, ACTA1, Rcan1.4, and β-MHC, detected by qRT-PCR in the heart tissues of the two groups of HFpEF mice are as follows Fig.26 As shown, refer to Fig.15From Figures A to E in Figure 1, the mRNA levels of five genes, ANP, BNP, ACTA1, Rcan1.4, and β-MHC, in the heart tissues of mice in the CTRP9 229-333 group were significantly lower than those in the control group. It can be seen that the CTRP9 229-333 polypeptide effectively inhibited the expression of cardiac hypertrophy genes, thereby inhibiting the hypertrophy of cardiomyocytes in HFpEF mice.

[0319] In summary, CTRP9 229-333 peptide can reduce the heart weight and lung weight of HFpEF mice, improve their exercise capacity, and improve their diastolic function. It can be seen that CTRP9 229-333 peptide has a certain therapeutic effect on HFpEF mice and inhibits myocardial remodeling caused by heart failure.

[0320] In addition, CTRP9 229-333 peptide can inhibit cardiac fibrosis in HFpEF mice by inhibiting the expression of cardiac fibrosis genes, and inhibit cardiomyocyte hypertrophy in HFpEF mice by inhibiting the expression of cardiac hypertrophy genes. Since the main pathological mechanisms of myocardial remodeling include myocardial fibrosis caused by fibroblast proliferation and extracellular matrix (ECM) reorganization, and myocardial hypertrophy caused by reduced cardiac contractility through mitochondrial dysfunction and metabolic abnormalities, and CTRP9 229-333 peptide can inhibit the expression of cardiac fibrosis genes and hypertrophy genes, it further shows that CTRP9 229-333 peptide can inhibit myocardial remodeling.

[0321] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the marketplace, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein. The scope of the present disclosure is defined by the appended claims.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is: NEFNHYDTAAGKFTCHIAGVYYFTYHITVFSRNVQVSLVKNGV KILHTKDAYMSSEDQASGGIVLQLKLGDEVWLQVTGGERFNGLFA DEDDDTTFTGFLLFSSP.

2. The polypeptide according to claim 1, characterized in that The polypeptide is obtained by the following method: Step a: co-incubate the recombinant protein CTRP9 with Cathepsin G protein to obtain the experimental group sample; mix the recombinant protein CTRP9 with physiological saline to obtain the control group sample; Step b: Perform quantitative electrophoresis on the two groups of samples, compare the difference bands between the two groups of samples after staining, and cut the difference band near the molecular weight of 17KDa in the experimental group; Step c: performing mass spectrometry detection on the differential bands of the experimental group to obtain the amino acid sequence of the polypeptide; Step d: synthesizing the polypeptide based on the amino acid sequence.

3. Use of the polypeptide according to claim 1 in preparing products related to inhibiting myocardial remodeling.

4. The use according to claim 3, characterized in that: The related products are used to treat myocardial remodeling-related diseases, which at least include: stable angina pectoris, unstable angina pectoris, myocardial infarction, chronic ischemic heart disease, coronary artery spasm heart disease, and myocarditis.

5. The use according to claim 4, characterized in that: The myocardial remodeling-related diseases also include: diabetes combined with myocardial ischemia-reperfusion.

6. The use according to claim 4, characterized in that: The myocardial remodeling-related diseases also include: heart failure with preserved ejection fraction.

7. The use according to claim 3, characterized in that: The related products are used for inhibiting myocardial hypertrophy and cardiac fibrosis.

8. The use according to claim 3, characterized in that: The related products are used for inhibiting myocardial cell apoptosis.

9. The use according to claim 3, characterized in that: The related products are drugs or pharmaceutical compositions; the drugs or pharmaceutical compositions also include diluents, adjuvants and / or carriers that can be used in the pharmaceutical field.

10. The use according to claim 3, characterized in that: The related products are foods, health products, enteral nutrition preparations, veterinary drugs or feed additives.

Images