Application of guanylate binding protein 5 in preparation of medicine for preventing, relieving or treating cardiac hypertrophy and fibrosis

Abstract

The invention discloses an application of guanylate binding protein 5 (GBP5) in preparation of a medicine for preventing, relieving or treating cardiac hypertrophy and fibrosis, in particular to a medicine for preventing, relieving or treating cardiac hypertrophy and fibrosis. GBP5 transgenic mice and non-transgenic mice are selected for testing, each mouse is divided into a false operation group and an operation group, aortic arch constriction operation is performed on the operation group, aortic arch constriction is not performed on the false operation group, and then cardiac hypertrophy, fibrosis and cardiac functions of the mice in the false operation group and the operation group are determined, so that the cardiac function of the mice in the false operation group and the operation group is determined. The influence of GBP5 gene overexpression on cardiac hypertrophy induced by aortic arch constriction is researched. Results show that the over-expressed GBP5 gene can significantly inhibit cardiac hypertrophy and fibrosis and protect cardiac functions, so that GBP5 can be used for preparing drugs for preventing, relieving or treating cardiac hypertrophy and fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of guanine nucleotide binding protein 5 in the preparation of a medicament for preventing, alleviating or treating cardiac hypertrophy and fibrosis. The present application belongs to the technical field of medicine. BACKGROUND

[0002] Hypertrophic Cardiomyopathy (HCM) is a genetic heart disease, which refers to the pathological condition that the myocardial cells are stimulated excessively, resulting in changes in the function and structure of myocardial cells. The changes belong to pathological enlargement, accompanied by up-regulation of various gene expressions such as atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), B-type natriuretic peptide (BNP), and down-regulation of functional genes such as α-MHC, sarcoplasmic reticulum Ca 2＋ ATPase 2a (SERCA2a). HCM is a cardiovascular disease that exists universally in the middle-aged and elderly population, which can cause a series of heart problems, such as left ventricular outflow obstruction, myocardial fibrosis, myocardial ischemia, ventricular remodeling, mitral regurgitation, syncope, cardiac pump dysfunction, and may develop into dilated cardiomyopathy, heart failure, arrhythmia and sudden death.

[0003] At present, the research on pathological myocardial hypertrophy has the following types:

[0004] (1) The molecular regulation mechanism of myocardial hypertrophy induced by angiotensin II (Ang II) was revealed by transcriptome sequencing analysis. The study found that the gene expression of the Ang II-induced myocardial hypertrophy model changed very obviously at the 10th day, including the triggering of inflammatory response, the up-regulation of fibrosis and apoptosis-related genes, and the down-regulation of energy metabolism-related genes. The related drug therapies for treating the disease at present are: renin-angiotensin-aldosterone system, β-adrenergic receptor, calcium channel and SGLT2 inhibitor. If we want to cure myocardial hypertrophy fundamentally, we cannot rely only on inhibiting the growth of myocardial cells, but also need to reduce heart rate and reduce the load of the heart to solve the problem of myocardial hypertrophy. Moreover, these drugs usually have many side effects, so we must solve the problem from the root.

[0005] (2) In a comprehensive review of recent advances in the diagnosis and treatment of hypertrophic cardiomyopathy (HCM), a cardiac myosin inhibitor, aficamten (CK-274), was mentioned as having shown improvement in symptomatic HCM patients in the SEQUOIA-HCM and REDWOOD-HCM trials [Advancements in the Diagnosis and Treatment of Hypertrophic Cardiomyopathy: A Comprehensive Review[J]. Journal of Cardiovascular Development and Disease, 2024, 11(9): 290. DOI: 10.3390 / jcdd11090290.]. Its mechanism of action is to reduce the interaction between the myosin head and actin filaments to reduce myocardial contractility, thereby reducing the increase in cardiac load caused by myocardial hypertrophy. After checking the literature, cardiac myosin inhibitors (such as aficamten) have been proven to be safe, effective, and feasible, and can be used as second-line treatment for symptomatic obstructive hypertrophic cardiomyopathy (HCM), and with the development of medical progress, cardiac myosin inhibitors may gradually replace the current standard treatment for HCM and change the trajectory and natural progression of the disease [The clinical utility of cardiac myosin inhibitors for the management of hypertrophic cardiomyopathy: a scoping review[J]. Journal of Cardiovascular Development and Disease, 2024, DOI: 10.1007 / s10741-024-10476-w.]. However, its high cost and price limit the accessibility of this drug to the general patient population. In addition, because they can affect left ventricular function, they can also cause iatrogenic heart failure, so close monitoring of the patient's physical condition during the use of this drug is required, and it is reversible after stopping. There is still a lack of research literature on this new type of drug involving cardiac myosin inhibitors, and there are few clinical cases, and its long-term effectiveness and safety need to be further studied.Other treatment methods, such as drug therapy (including beta-blockers, calcium channel blockers, and antiarrhythmic drugs), alcohol septal ablation (ASA), and radiofrequency ablation (RFA), are also discussed in this review [Advancements in the Diagnosis and Treatment of Hypertrophic Cardiomyopathy: A Comprehensive Review[J]. Journal of Cardiovascular Development and Disease, 2024, 11(9): 290. DOI: 10.3390 / jcdd11090290. ]. These methods aim to alleviate symptoms, improve heart function, and reduce cardiovascular disease risk events.

[0006] (3) New therapeutic targets for myocardial hypertrophy, including blocking of cell surface receptors and signaling pathways, and histone deacetylase (HDAC) inhibitors, aim to inhibit the abnormal growth of cardiomyocytes to treat HCM [Winkle AJ, Nassal D M, Shaheen R, et al. Emerging therapeutic targets for cardiac hypertrophy[J]. Expert Opinion on Therapeutic Targets, 2022, 26(1): 29-40. DOI: 10.1080 / 14728222.2022.2031974]. There are many technical shortcomings in the research of this technology, such as uncontrollable cell metabolism and signaling pathways, unclear specific mechanisms, uncertain specific treatment plans, unclear long-term efficacy, low reproducibility probability, and insufficient precision. These shortcomings limit the investment of researchers in treatment strategies for myocardial hypertrophy.

[0007] In summary, these treatment strategies intervene in the abnormal growth of cardiomyocytes and reduce the load on the heart through different mechanisms to delay or improve heart function in HCM patients. These studies will become the basis for curing HCM, and their shortcomings and research methods are worth learning and understanding.

[0008] Guanylate Binding Protein 5 (GBP5) is a very important protein in the Guanylate Binding Protein (GBP) family, which can participate in human immune regulation, antiviral response and inflammatory response, and enhance human immunity through mechanisms such as interaction with inflammasome components, affecting the maturation and release of inflammatory factors, and regulating pyroptosis. However, it is not clear whether GBP5 has a role in heart disease, especially in the pathological process of myocardial hypertrophy. SUMMARY

[0009] To solve the defects and deficiencies of the prior art in the clinical prevention and treatment of pathological myocardial hypertrophy disease, the purpose of the present application is to determine the mutual relationship between the expression of GBP5 gene and pathological myocardial hypertrophy. A new use for treating pathological myocardial hypertrophy is provided, and the GBP5 gene is applied to the treatment of pathological myocardial hypertrophy.

[0010] In order to achieve the above-mentioned purpose, the following technical means are adopted in the present application:

[0011] (1) GBP5 gene knockout significantly promotes myocardial hypertrophy, fibrosis and worsens cardiac function

[0012] In the present application, wild type mice and GBP5 gene knockout mice are selected for testing, and each type of mouse is divided into a sham operation group and a surgery group, with 10 mice in each group. The surgery group is given aortic arch constriction surgery, and the sham operation group is not given aortic arch constriction. Then, the myocardial hypertrophy, fibrosis and cardiac function of the mice in each group of the sham operation group and the surgery group are measured to study the effect of GBP5 gene knockout on myocardial hypertrophy induced by aortic arch constriction. The results show that knockout of the GBP5 gene significantly worsens myocardial hypertrophy, fibrosis and cardiac function.

[0013] (2) Overexpression of GBP5 gene significantly inhibits myocardial hypertrophy and fibrosis and improves cardiac function

[0014] In the present application, heart-specific GBP5 transgenic mice and non-transgenic mice are selected for testing, and each type of mouse is divided into a sham operation group and a surgery group, with 10 mice in each group. The surgery group is given aortic arch constriction surgery, and the sham operation group is not given aortic arch constriction. Then, the myocardial hypertrophy, fibrosis and cardiac function of the mice in each group of the sham operation group and the surgery group are measured to study the effect of GBP5 gene overexpression on myocardial hypertrophy induced by aortic arch constriction. The results show that overexpression of the GBP5 gene significantly inhibits myocardial hypertrophy and fibrosis and protects cardiac function.

[0015] On the basis of the above-mentioned research, the application provides application of guanosine nucleotide binding protein 5 in preparation of a drug for preventing, alleviating or treating myocardial hypertrophy and fibrosis.

[0016] Preferably, the amino acid sequence of the guanosine nucleotide binding protein 5 is shown as SEQ ID NO. 1.

[0017] Preferably, the myocardial hypertrophy is pathological myocardial hypertrophy.

[0018] Further, the application also provides application of a coding sequence of guanosine nucleotide binding protein 5 in preparation of a drug for preventing, alleviating or treating myocardial hypertrophy and fibrosis.

[0019] Preferably, the coding sequence of the guanosine nucleotide binding protein 5 is shown as SEQ ID NO. 2.

[0020] Preferably, the myocardial hypertrophy is pathological myocardial hypertrophy.

[0021] Further, the application also provides application of a vector containing the coding sequence of guanosine nucleotide binding protein 5 in preparation of a drug for preventing, alleviating or treating myocardial hypertrophy and fibrosis.

[0022] Preferably, the coding sequence of the guanosine nucleotide binding protein 5 is shown as SEQ ID NO. 2.

[0023] Preferably, the vector is an adenovirus expression vector for overexpressing guanosine nucleotide binding protein 5.

[0024] Preferably, the myocardial hypertrophy is pathological myocardial hypertrophy.

[0025] Compared with the prior art, the application has the following beneficial effects:

[0026] (1) The application discovers a new function of GBP5, that is, GBP5 has the effects of inhibiting myocardial hypertrophy and fibrosis and improving heart function.

[0027] (2) Based on the effect of GBP5 on inhibiting occurrence of myocardial hypertrophy disease, the GBP5 can be used for preparing a drug for preventing and treating myocardial hypertrophy disease. DETAILED DESCRIPTION

[0028] Figure 1 is a representative graph of GBP5 Western Blot and a statistical graph of TAC model mice and myocardial tissues of hypertrophy patients and normal donors, a representative graph of GBP5 Western Blot and a statistical graph of PE treatment of primary mouse myocardial cells in vitro and a qRT-PCR statistical graph;

[0029] Among them, (A) representative Western Blot and statistical graphs of GBP5 in myocardial tissue from hypertrophic patients and normal donors (n = 3); (B) representative Western Blot and statistical graphs of GBP5 and qRT-PCR in heart tissue from TAC model mice (n = 6); (C) representative Western Blot and statistical graphs of GBP5 and qRT-PCR in primary neonatal mouse cardiomyocytes treated with PE in vitro (n = 6). All data were analyzed using Mean ± SEM, *P < 0.05;

[0030] Figure 2 Systemic GBP5 knockout exacerbates TAC-induced cardiac remodeling;

[0031] Among them, (A) TAC model mice, systemic GBP5 knockout mice (GBP5 - / - (n = 6) Gross representation of the heart in WT mice, scale bar: 2 mm; (BC) Statistical plots of heart weight ratio and heart weight-to-tibia length ratio in systemic GBP5 knockout mice and WT mice in TAC model mice (n = 6); (DF, HI) Representative and statistical plots of HE, PSR, Masson, and WGA in the heart of systemic GBP5 knockout mice and WT mice in TAC model mice (n = 6), scale bar: 1 mm or 40 µm; (J, K) Statistical plots of EF and FS (n = 6); (G, MO, RT) Representative and statistical plots of echocardiograms in systemic GBP5 knockout mice and WT mice in TAC model mice (n = 6); (L, PQ, UV) qRT-PCR statistical plots of cardiac hypertrophy and fibrosis in systemic GBP5 knockout mice and WT mice in TAC model mice (n = 6); All data are expressed as mean ± SEM, *P < 0.05, *P < 0.05, **P < 0.05. 0.01, ***P <0.001, ****P < 0.0001;

[0032] Figure 3 The result of cardiomyocyte-specific GBP5 knockout exacerbating TAC-induced cardiac remodeling is that...

[0033] Note: (A) In TAC model mice, GBP5 cKO Rats and GBP5 f / f Gross representation of a mouse heart, scale bar: 2 mm; (BC) TAC model mice, GBP5 cKO Rats and GBP5 f / f Statistical plot of heart-weight to tibia length ratio and heart-weight ratio (n = 4-5); In (DM)TAC model mice, GBP5 cKO Rats and GBP5 f / fRepresentative images of heart ultrasound, EF, FS, and individual ventricular wall thickness and ventricular diameter (n = 4-5); (N-Q) In TAC mice, GBP5 cKO mice and GBP5 f / f Representative images of heart HE, PSR, Masson and WGA and statistical graphs, scale bar: 1 mm or 40 pm (n = 3-4); (R, S) ANP, BNP and b-MHC and degree of myocardial fibrosis qRT-PCR results; (T, U) Representative images of heart ANP Western Blot and statistical graphs (n = 3); all data using mean ± SEM, *P < 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0034] Figure 4 is the result of myocardial cell-specific GBP5 overexpression alleviating TAC-induced cardiac remodeling;

[0035] Note: (A) In TAC mice, GBP5-Tg mice and GBP5 cki / cki Representative images of mouse heart gross, scale bar: 2 mm; (B-C) In TAC mice, GBP5-Tg mice and GBP5 cki / cki Statistical graphs of heart weight and heart weight / tibia length (n = 4-5); (D-G, I-J, N-O) In TAC mice, GBP5-Tg mice and GBP5 cki / cki Representative images of mouse heart ultrasound and EF, FS, and individual ventricular wall thickness and ventricular diameter statistical graphs (n = 4-5) (H, L, M, K, P) In TAC mice, GBP5-Tg mice and GBP5 cki / cki Representative images of mouse heart HE, PSR, Masson and WGA and statistical graphs, scale bar: 1 mm or 40 pm (n = 3); (Q-R) In TAC mice, GBP5-Tg mice and GBP5 cki / cki Representative images of mouse heart ANP Western Blot and statistical graphs (n = 3); (S-T) In TAC mice, GBP5-Tg mice and GBP5 cki / cki Representative images and statistical graphs of mouse heart hypertrophy and fibrosis indicators (n = 3-5), all data using mean ± SEM, *P < 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0036] Figure 5 is the result of GBP5 alleviating PE-induced myocardial hypertrophy;

[0037] Note: (A, C) Western Blot representative graph and statistics of ANP in PE-treated primary rat cardiomyocytes (n = 3-6); (B, D) Immunofluorescence representative graph and statistics of PE-treated primary rat cardiomyocytes (n = 6), scale bar: 20 pm, all data using Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0038] Figure 6 is knockdown of GBP5 to promote the results of PE-induced cardiac glycolysis;

[0039] Note: (A) RNA-seq differential genes and KEGG analysis of PE-treated primary rat cardiomyocytes (n = 4); (B-C) Seahorse glycolysis stress test chart and statistics (n = 6); (D-G) Western Blot representative graph of lactate antibody in each group of cells; (D-G) Western Blot representative graph and statistics of H3K18 lactate in each group of cells (n = 3); all data using Mean ± SEM, *P < 0.05. DETAILED DESCRIPTION

[0040] The present application is further illustrated by the following examples, which are intended to better illustrate the research content of the present application and not to limit the protection scope of the present application. In the following examples, except for special cases, they are the conventional experimental methods and operation steps in the art.

[0041] Experimental animals and feeding:

[0042] Experimental animals: systemic GBP5 knockout mice (GBP5 KO or GBP5 - / - ), purchased from RIKEN Company, Japan, item number: RIKEN01772; myocardial cell-specific GBP5 knockout mice (GBP5 cKO ), myocardial cell overexpression of GBP5 mice (GBP5 cki / cki ), systemic GBP5 gene overexpression mice (GBP5-Tg) purchased from Saiye Biotechnology Co., Ltd., non-transgenic mice (NTG), same-age littermate control non-transgenic mice.

[0043] Example 1: Myocardial hypertrophy model obtained

[0044] 1. Experimental animal grouping: male background C57BL / 6 wild-type mice (WT), GBP5 knockout mice (GBP5 KO), myocardial hypertrophy model was established by aortic constriction (TAC). Randomly divided into 8 groups, grouping as follows: C57BL / 6 background wild type mice sham operation group (WT Sham) and TAC operation group (WT TAC), GBP5 gene knockout mice sham operation group (GBP5 KO Sham) and TAC operation group (GBP5 KO TAC), non-transgenic mice sham operation group (NTG Sham) and TAC operation group (NTG TAC).

[0045] 2. Myocardial hypertrophy model adopts aortic arch constriction (TAC) operation, model operation process:

[0046] 2.1 Preoperative preparation

[0047] (1) Anesthesia: First, weigh the mouse, calculate the amount of anesthetic (3% sodium pentobarbital) needed according to 90mg / kg body weight, inject intraperitoneally, and record the injection time point. The success criteria for anesthesia are that there is no obvious reaction when the tail and toes are clamped, and the mouse is in good condition (usually no obvious reaction after injection for about 10 min, and the mouse has a reaction when the toes are clamped after anesthesia for about 50 min, and the optimal operation time is about 30 min after anesthesia).

[0048] (2) Preparation of the operation area: Shave the skin of the left chest, left side of the chest and left forelimb of the mouse. After shaving, use a wet gauze to wipe the operation area to remove the mouse hair, so as not to affect the surgical field.

[0049] (3) Tracheal intubation: Use a rubber band to fix the mouse's upper incisors on the inclined surface of the V-shaped plate, and quickly insert the tracheal tube through the glottis into the trachea. Then, place the mouse in a right lateral position on a heating pad (the heating pad needs to be preheated), then connect the tracheal tube to the respirator, and fix the mouse. If the mouse's chest rises and falls in sync with the respirator's frequency, it means the tracheal intubation is successful.

[0050] 2.2 Aortic arch branch ligation (TAC)

[0051] The right lateral decubitus position was taken, and the left forelimb of the mouse was placed above the right forelimb, and the two forelimbs were fixed with medical tape. A cotton swab was placed under the right chest, the chest was elevated, and the skin of the surgical area was disinfected with iodine and 75% alcohol by volume in turn. The left hand held an ophthalmic forceps to pinch the left chest skin, and the right hand held an ophthalmic scissors to cut the skin about 1 cm, and then the muscles and soft tissues were separated in turn, and the thoracic cavity was opened at the level of the 2-3 ribs, the left lung was slightly probed with a cotton swab, the descending branch of the aortic arch was isolated, a 7-0 surgical suture was passed through the blood vessel, and a 26G (25.0-27.5g mouse) or 27G (23.5-25.0g) syringe needle was placed parallel above the blood vessel, the blood vessel and the needle were ligated together, and then the needle was withdrawn to achieve the corresponding degree of blood vessel constriction. After ligation, the incision was sutured in turn, the thoracic cavity was closed, a syringe was inserted into the thoracic cavity from the suture opening and 1cc gas was withdrawn to restore the negative pressure in the thoracic cavity, and then the syringe was pulled out and the skin incision was quickly sutured. The sham group (Sham) was only threaded without ligation after the descending branch of the aorta was isolated, and the rest of the steps were the same as the myocardial hypertrophy (TAC) model group.

[0052] 2.3 Postoperative care

[0053] After the descending branch of the aortic arch was ligated, the mouse was placed in a feeding cage containing high-temperature sterilized bedding, feed and drinking water in the feeding room for continued feeding and observation. The GBP5 gene knockout mice and wild type mice were detected for various indicators at 4 weeks after the operation.

[0054] Example 2 GBP5 protein expression and mRNA expression in heart tissue of patients with hypertrophic cardiomyopathy, TAC model mice and PE-treated neonatal rat primary myocardial cells

[0055] The human hypertrophic cardiomyopathy (HCM) tissue was derived from hypertrophic cardiomyopathy patients, and the normal myocardial tissue (Ctrl) was derived from normal donors. The information of normal donors and patients is shown in Table 1. The GBP5 protein expression level of myocardial tissue cells derived from hypertrophic cardiomyopathy patients and myocardial tissue cells derived from normal donors was detected.

[0056] Table 1. Information table of normal donors and patients

[0057]

[0058] TAC mice were sacrificed 4 weeks after feeding, and heart tissue was collected for protein and RNA extraction. Protein was extracted using RIPA lysis buffer, and SDS-PAGE and Western blot analysis was performed to detect the expression levels of GBP5 and the myocardial hypertrophy marker ANP, with GAPDH as an internal control. RNA was extracted using Trizol, reverse transcribed into cDNA, and then subjected to real-time quantitative PCR (qRT-PCR) to detect the level of GBP5 mRNA, with the results analyzed using the relative quantification method. At the same time, primary myocardial cell experiments used 1-3 day old neonatal mouse ventricular tissue. After enzymatic digestion to separate the myocardial cells and remove fibroblasts, the cells were cultured adherently. The experimental group was treated with 50 μM phenylephrine (PE) for 48 hours to induce cell hypertrophy, and the control group was not treated. After treatment, the cells were also subjected to protein and RNA extraction, and the expression of GBP5 and ANP was detected by Western blot and qRT-PCR, respectively.

[0059] Results: As shown in Figure 1 Figure 1, the level of GBP5 protein was significantly increased in the hearts of patients with hypertrophic cardiomyopathy Figure 1 A). In TAC model mice, the levels of GBP5 protein and mRNA were significantly increased Figure 1 B). In the in vitro PE-treated primary mouse myocardial cell hypertrophy model, the levels of GBP5 protein and mRNA were significantly increased Figure 1 C). The above results indicate that the expression of GBP5 is significantly increased in various hypertrophic cardiomyopathy models.

[0060] The above two models verify the changes in GBP5 expression under myocardial hypertrophy from in vivo and in vitro levels, aiming to reveal its potential regulatory role in cardiac remodeling.

[0061] Example 3 Detection of mouse heart function, myocardial hypertrophy, and fibrosis

[0062] 1. Ultrasound detection of heart function in myocardial hypertrophy model mice

[0063] 1.1 Preliminary preparation

[0064] (1) Anesthesia machine preparation: first connect the oxygen cylinder to the gas inlet interface on the anesthesia machine, then unscrew the drug addition port seal cover on the anesthesia machine, quickly add isoflurane to the safe scale, and then tighten the seal cover. Unscrew the total valve on the oxygen cylinder, adjust the rotation knob of the flow control valve, and maintain the outlet gas pressure at 0.2-0.3 mPa.

[0065] (2) Preparation of mice to be tested: after the mice to be tested were anesthetized with isoflurane, the left chest area was shaved, and the treated mouse head was inserted into the anesthesia conduit sleeve head. Maintain the mouse in a stable anesthetized state with 1.5-2.0% isoflurane.

[0066] 1.2 Heart function detection

[0067] The mice were taken left lateral decubitus or supine position, and the ultrasonic coupling agent (Tianjin Chengxin Company) was uniformly applied in the shaved area. A high-frequency ultrasonic diagnostic instrument was used, the frequency was 15 MHz, the standard left ventricular papillary muscle short axis section was selected, and the left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), ejection fraction (EF) and short axis shortening rate (FS) were measured. In this embodiment, M-mode echocardiogram was used to detect and evaluate myocardial hypertrophy and heart function.

[0068] 2, Myocardial hypertrophy and fibrosis detection of myocardial hypertrophy model of mice

[0069] 2.1 Sampling

[0070] (1) Preliminary work: Prepare a urine cup containing 20 mL of 10% formaldehyde in advance, and label it (mouse number, group, operation type and sampling date). Place a culture dish filled with 10% KCl solution at the sampling site. Turn on the analytical balance and adjust it to zero for standby. Then weigh the dead mouse.

[0071] (2) Sampling: Hold the blood vessel pedicle below the auricle with an ophthalmic curved forceps, cut off the heart, and quickly place it in a 10% KCl solution. After the heart stops beating in diastole, place it on sterile gauze, gently squeeze the liquid in the heart cavity, and after wiping the surface liquid, weigh and record, and place the heart in the corresponding urine cup. After 48 hours of fixation, it is used for pathological detection.

[0072] (3) Related measurement and calculation: Take out the mouse lung, filter paper after trimming, weigh and record. Cut the skin at the tibia of the mouse hind limb, measure and record the tibia length. Calculate the ratio of heart weight to body weight (HW / BW) and the ratio of heart weight to tibia length (HW / TL).

[0073] 2.2 Pathological detection

[0074] 2.2.1 Preparation of paraffin specimen section

[0075] The main operation procedures include trimming the heart → embedding frame treatment → water flushing → dehydration → transparency → wax immersion → embedding → sectioning → spreading → drying or baking for standby.

[0076] 2.2.2 Hematoxylin-eosin (HE) staining

[0077] The main steps are as follows: baking at 55℃ for 30 min → xylene for 5 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → double distilled water for 1 min → hematoxylin solution (Zhuhai Beso, BA-4021) for 5 min → water wash for 1 min → 1% hydrochloric acid alcohol (take 3 mL of concentrated hydrochloric acid and 297 mL of 70% alcohol and mix thoroughly) for 1-3 s → water wash for 1 min → Scott's solution (0.35 g of sodium bicarbonate and 2 g of magnesium sulfate heptahydrate dissolved in 100 mL of distilled water) for 1 min → water wash for 1 min → eosin solution (Zhuhai Beso, BA-4024) for 3-5 min → wash away the floating color with distilled water → 70% alcohol for 1 s → 95% alcohol for 1 s → 100% alcohol for 30 s, 3 times → xylene for 2 min, 3 times → immediately seal the slide while the xylene is still wet → dry in a fume hood and take pictures under a microscope.

[0078] HE staining image statistics: Select more than 3 cells with clear boundaries and nuclei roughly in the center from each image, and circle the cell area using Image-Pro Plus 6.0 software.

[0079] 2.2.3 Sirius Red (PSR) Staining

[0080] The main steps are as follows: baking at 55℃ for 30 min → xylene for 2 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → rinsing with running water for 10 min → double-distilled water for 1 min → 0.2% phosphomolybdic acid for 2 min → 0.1% Sirius red picric acid solution dropped onto the tissue, staining in a humidified chamber for 90 min → removing residual solution → 0.01N hydrochloric acid for 4 s → 70% alcohol once → 90% alcohol once → 100% alcohol for 30 s, 3 times → xylene for 2 min, 3 times → immediately cover with a slide while the xylene is still wet, and take pictures under a microscope.

[0081] Myocardial tissue is composed of cardiomyocytes and interstitial tissue. The heart is a terminally differentiated organ; cardiomyocytes have lost their proliferative capacity. Various physiological or pathological stimuli can only cause an increase in the size of individual cells, not a proliferation of their numbers. Therefore, the pathophysiology of myocardial hypertrophy is mainly characterized by increased cardiomyocyte size, increased number of sarcomeres, disordered cell arrangement, and interstitial changes including the proliferation and transformation of myocardial fibroblasts, increased collagen fiber density, increased collagen secretion, and an imbalance in collagen ratios.

[0082] result:

[0083] Figure 2Figure 6. Heart morphology, echocardiography, and qRT-PCR analysis of mice with systemic knockout of GBP5. A, B, C, D, E, H, U, V) Heart morphology, heart weight / body weight ratio, and heart weight / tibia length ratio of mice with systemic knockout of GBP5. F, I, L, P, Q) HE, PSR, Masson, and WGA staining of mice with systemic knockout of GBP5. G, J, K, M, N, O, R, S, T) Echocardiography and qRT-PCR analysis of mice with systemic knockout of GBP5. Figure 2 A, B, C, D, E, H, U, V). After WGA staining, it was found that TAC significantly increased the area of cardiomyocytes, especially in the group of mice with systemic knockout of GBP5. RT-PCR results also showed that the expression of ANP, BNP, and β-MHC (markers of myocardial hypertrophy) was significantly increased in mice with systemic knockout of GBP5 after TAC, further indicating that knockout of GBP5 significantly increased TAC-induced myocardial hypertrophy. Figure 2 F, I, L, P, Q). After WGA staining, it was found that TAC significantly increased the area of cardiomyocytes, especially in the group of mice with systemic knockout of GBP5. RT-PCR results also showed that the expression of ANP, BNP, and β-MHC (markers of myocardial hypertrophy) was significantly increased in mice with systemic knockout of GBP5 after TAC, further indicating that knockout of GBP5 significantly increased TAC-induced myocardial hypertrophy. - / - +sham、WT + TAC、GBP5 - / - + TAC, and observed the heart function after 6 weeks. It was found that the ejection fraction and fractional shortening of the TAC group were significantly lower than those of the sham group, and the indicators of myocardial hypertrophy in this group were significantly increased. Figure 2 G, J, K, M, N, O, R, S, T).

[0084] To verify whether the GBP5 in cardiomyocytes plays a role in myocardial hypertrophy, we constructed mice with myocardial-specific knockout of GBP5, and the control mice and mice with myocardial-specific knockout of GBP5 were subjected to TAC and sham treatment, respectively. Figure 3is the result of myocardial cell-specific GBP5 knockout aggravating TAC-induced cardiac remodeling. From the heart morphology map, the myocardial-specific GBP5 knockout mice in the TAC group were compared with other groups, which proved that GBP5 knockout worsened TAC-induced myocardial hypertrophy, and the HW / TL and HW / BW ratios were significantly higher than those of other groups. Figure 3 A, B, C); HE: increased cross-sectional area of myocardial cells, WGA staining further confirmed myocardial hypertrophy; echocardiography proved that cardiac function was significantly reduced Figure 3 D, E, F, G, H, I, K, L); PSR, Masson showed that the degree of collagen deposition and fibrosis was seriously aggravated Figure 3 O, P, Q, N, J, M); qRT-PCR results showed that the myocardial hypertrophy-related indicators ANP, BNP, and β-MHC and the degree of myocardial fibrosis in myocardial-specific knockout mice after TAC were significantly higher than those of other groups Figure 3 R, S), and the WB results also showed that myocardial-specific knockout of GBP5 could induce TAC to significantly increase the expression level of ANP protein Figure 3 T, U). The above experiments further confirmed that the specific knockout of GBP5 indeed aggravated TAC-induced heart failure, myocardial hypertrophy, and myocardial fibrosis. The above results showed that myocardial-specific knockout of GBP5 significantly increased TAC-induced myocardial hypertrophy.

[0085] Figure 4 is the TAC model mouse, GBP5-Tg mouse and GBP5 cki / cki Heart morphology map, heart weight to tibia length ratio and heart weight to body weight ratio statistics (n = 4-5); echocardiogram and EF, FS, and each ventricular wall thickness and ventricular diameter statistics (n = 4-5); HE, PSR, Masson and WGA representative diagram and statistics; heart ANP Western Blot representative diagram and statistics (n = 3); heart hypertrophy and fibrosis index representative diagram and qRT-PCR statistics (n = 3-5). The heart morphology map shows that overexpression of GBP5 can significantly alleviate TAC-induced hypertrophy. Heart weight to tibia length ratio, heart weight to body weight ratio also found that overexpression of GBP5 can significantly alleviate myocardial hypertrophy (Fig. 4A, B, C). Echocardiogram results show that compared with the GBP5 cki / cki + TAC group, the heart function of the GBP5-Tg + TAC group is improved, the ejection fraction of the GBP5 overexpression mouse heart, the short axis shortening rate decreases, the left ventricular internal diameter diastolic and systolic degree increases, and the left ventricular myocardial weight significantly decreases (Fig. 4D, E, F, G, I, J, N, O). HE staining shows that the cross-sectional area of the heart is reduced Figure 4 H). The experimental results show that the Masson results show that compared with the GBP5 cki / cki+ TAC group (Fig. 4B). The collagen deposition and fibrosis were significantly reduced in the GBP5-Tg + TAC group compared with the TAC group (Fig. 4B). Figure 4 L, P, T); WGA results showed that, compared with the WT + TAC group, the GBP5 cki / cki + TAC group (Fig. 4B). The collagen deposition and fibrosis were significantly reduced in the GBP5-Tg + TAC group compared with the TAC group (Fig. 4B). Figure 5 M, K). qRT-PCR results showed that, compared with the WT + TAC group, the GBP5 cki / cki + TAC group (Fig. 4B). The collagen deposition and fibrosis were significantly reduced in the GBP5-Tg + TAC group compared with the TAC group (Fig. 4B). Figure 4 S), which indicated that overexpression of GBP5 caused pathological cardiac hypertrophy in the pressure overload model (Fig. 4C). WB results also showed that overexpression of GBP5 could significantly reduce the protein expression level significantly increased by TAC (Fig. 4C). Figure 4 Q, R).

[0086] Example 3 Effects of interference (Adsh GBP5) and overexpression (Ad GBP5) adenovirus of GBP5 on PE-stimulated hypertrophy of primary myocardial cells

[0087] 1. Culture of primary neonatal Kunming mouse milk mouse myocardial cells

[0088] (1) Eight neonatal 1-day Kunming mouse milk mice were sterilized with 75% alcohol below the neck, and the heart was taken out with ophthalmic scissors and microforceps and placed in a glass dish containing 10 mL of DMEM / F12 liquid. Repeat the above process with another one.

[0089] (2) Wash the heart with DMEM / F12 medium, and cut the heart into 1-2 mm pieces. Transfer to a serum bottle with a rotor, aspirate the DMEM / F12, and add trypsin digestion solution. The rotation speed is 120 r / min, and the digestion time is 15 min. Rest for a few seconds, and discard the supernatant.

[0090] (3) Add trypsin digestion solution, rotate at 120 r / min for 15 min. Rest for a few seconds, aspirate the supernatant, terminate the digestion with 20% calf serum DMEM / F12 medium, and store in a 4°C refrigerator. Repeat the step for several cycles. When taking the supernatant, try to take it all out, and when the tissue pieces become white and significantly smaller, stop the digestion.

[0091] (4) Centrifuge the collected myocardial cell suspension at 1500 rpm for 8 min, discard the supernatant. Add an appropriate amount of medium to the centrifuge tube, gently blow and resuspend the cells, concentrate them into one 50 mL centrifuge tube, and filter the cell suspension with a cell 40 μm filter.

[0092] (5) Cells were seeded in 100 mm dishes and allowed to adhere for 90 min. Non-adherent cells were removed by aspiration and filtration. Brdu was added to a final concentration of 0.1 mM based on the total volume of the cell suspension. After mixing, the cells were plated in 0.1% gelatin-coated dishes.

[0093] (6) Gently disperse the cells without vortexing. Incubate at 37°C, 5% CO2 for 48 hours. Wash once with PBS and change the medium.

[0094] 2. The effect of interference (Adsh GBP5) and overexpression (Ad GBP5) of adenovirus of GBP5 on the myocardial hypertrophy model induced by PE

[0095] Adenovirus vector containing Adsh GBP5 was purchased from Jiangsu Gencefe Biological Technology Co., Ltd. with the product number S10001-A. AdGFP (adenovirus vector containing GFP (green fluorescent protein)) and Ad GBP5 (adenovirus vector containing GFP-GBP5 (green fluorescent protein-GBP5 fusion protein)) were purchased from Shandong Wezhen Biological Technology Co., Ltd.

[0096] Adenovirus containing Adsh GBP5, adenovirus containing AdGFP and adenovirus containing Ad GBP5 were used to infect primary myocardial cells cultured for 3 days at 10 MOIs. After 12 hours, 1 μM phenylephrine (PE) or control PBS was used for stimulation for 48 hours, and then immunofluorescence test was performed.

[0097] 3. Real-time PCR:

[0098] High-speed centrifuge pre-cooling, 75% ethanol is configured in advance and pre-cooled, the whole process of extracting RNA is operated on the ice box; collect cells, wash twice with PBS buffer, add 1 mL Trizol lysis buffer to each sample, repeat blowing and beating, and stand for 5 min; add 200 µL chloroform, (add 100 µL to each well of the 12-well plate) upside down evenly, stand for 5 min, after standing, centrifuge at 4°C, 13300 rpm for 15 min, after centrifugation, absorb 500 µL supernatant (200 µL for cells) into a new tube, then add an equal volume of isopropanol 500 µL (200 µL for cells), mix well and stand for 20 min, at the same time, according to the required amount, prepare 75% alcohol (1 mL alcohol = 750 µL anhydrous ethanol + 250 µL DEPC water), mix well after preparation and reserve; after standing, centrifuge at 4°C for 12 min, discard the supernatant, add 1 mL of prepared 75% alcohol to each sample, shake to make the precipitate float, centrifuge at 4°C, 10600 rpm for 7 min, discard the supernatant, and invert the test tube to dry the precipitate into a transparent state, add 10 µL DEPC water to repeat blowing and beating (if the precipitate is relatively large, add 15 µL~20 µL DEPC water), use NanoDrop / Bioanalyzer to evaluate the concentration and integrity, record the concentration 1, concentration 2, concentration 3 of each sample, and calculate the average sample concentration and the amount of DEPC water, configure the system according to 2× SYBR Green, DEPC water, upstream primer F, downstream primer R, respectively 10 µL, 7 µL, 1 µL, 1 µL, sample RNA 1 µL (total system is 10 µL), blow and mix after configuration, use reverse transcription kit HiScript® III RT SuperMix for qPCR (+gDNAwiper) (company: Novozyme; article number: R323; specification: 100 rxns (20ul / rxn)) for transcription, the transcription program is reaction condition 94°C hot start for 10 min, 94°C for 15 s, 55°C for 1 s, 72°C for 20 s, 35 cycles. After transcription, measure the curve on the machine, all biological repeats are statistically analyzed within the group and appropriate statistical tests are performed to evaluate significance.

[0099] 4. RNA-seq

[0100] Total RNA was extracted from PE-treated and control primary neonatal rat cardiomyocytes using TRIzol or column kits, and the quality (RIN ≥ 7.0) was detected by NanoDrop and Bioanalyzer. The qualified samples were subjected to mRNA enrichment, fragmentation, reverse transcription, A addition, adapter ligation, and PCR amplification to construct the library (such as Illumina TruSeq), and then subjected to double-end sequencing on the Illumina NovaSeq platform. After quality control by fastp and alignment to the mouse reference genome by STAR, the sequencing data were counted by featureCounts and analyzed for differential expression by DESeq2 (|log2FC| > 1, FDR < 0.05), and then subjected to KEGG enrichment analysis by clusterProfiler to screen for sugar metabolism-related pathways (n = 4).

[0101] 5. Seahorse glycolysis stress test

[0102] Primary cardiomyocytes were seeded at 2-4 x 10 4 After adhesion, the medium was replaced with glucose-free assay medium, and the cells were equilibrated at 37°C in a non-CO2 environment for 1 hour. During the test, glucose (10 mM), oligomycin (1 µM), and 2-DG (50 mM) were injected in sequence, and the extracellular acidification rate (ECAR) was recorded in real time. The glycolysis rate, glycolysis capacity, and glycolysis reserve were analyzed, and the results were normalized by protein amount and subjected to statistical analysis (n = 6).

[0103] 6. Western blot experiment

[0104] Proteins were obtained by RIPA lysis or 0.4 N H2SO4 acid extraction, quantified by BCA, denatured with SDS buffer, and 20-40 µg of protein was separated by 12-15% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% BSA, the primary antibody (Pan-Kla, H3K18la, and Histone H3) was incubated at 4°C overnight, and the next day the HRP secondary antibody was incubated and developed by ECL. The band gray scale was analyzed by ImageJ, the target signal was normalized by Histone H3, and the average of three repeated experiments was taken (n = 3). In general, this method integrates transcriptomic, cell metabolic function determination, and protein modification level analysis to systematically evaluate the negative regulatory role of GBP5 in PE-induced enhancement of myocardial glycolysis and lactate modification.

[0105] Results:

[0106] Figure 5These are representative Western blotting and statistical images (n = 3-6) of ANP in primary neonatal rat cardiomyocytes treated with PE; and representative immunofluorescence images and statistical images (n = 6). After inducing GBP5 silencing in primary neonatal rat cardiomyocytes with norepinephrine (PE), Western blotting results showed that, compared to the control group, GBP5 gene silencing promoted the expression of the hypertrophy gene ANP, and GBP5 silencing significantly exacerbated PE-induced ANP expression. Immunofluorescence images showed that the cross-sectional area of ​​cardiomyocytes in the siGBP5 + PE group was significantly larger than that in other groups. These results indicate that GBP5 gene silencing exacerbates PE-induced cardiac hypertrophy (…). Figure 5 A, B).

[0107] In in vitro experiments, we treated primary neonatal rat cardiomyocytes with PE to investigate the role of GBP5 overexpression (oeGBP5) in myocardial hypertrophy. Our results showed that, compared with the oeNC + PE group, the oeGBP5 + PE group significantly reduced the cardiomyocyte hypertrophy marker ANP. Immunofluorescence assays showed that, compared with the oeNC + PE group, the oeGBP5 + PE group significantly reduced the cardiomyocyte area (…). Figure 5 C, D) The results above indicate that overexpression of GBP5 reduces myocardial hypertrophy caused by PE.

[0108] Figure 6 This includes RNA-seq and KEGG analysis of differentially expressed genes in PE-treated primary neonatal rat cardiomyocytes (n = 4); Seahorse glycolytic stress assay plots and statistical graphs (n = 6); Western blotting representations of lactated pan-antibodies in each cell group; and Western blotting representations and statistical graphs of H3K18 lactated cells in each cell group (n = 3). RNA-seq and KEGG analysis were performed on PE-treated primary neonatal rat cardiomyocytes. Figure 6 A), the results showed that differentially expressed genes were significantly enriched in glucose metabolism-related pathways; further analysis using the Seahorse glycolytic stress assay revealed that, compared with the control group, GBP5 knockdown significantly enhanced the glycolytic rate, glycolytic capacity, and glycolytic reserve of cells under PE stimulation. Figure 6 B, C); Western blot results showed that both the TAC model and PE treatment induced upregulation of pan-lactation in cardiomyocytes at the protein lactation level. Figure 6 D, E), and GBP5 knockdown further enhanced PE-induced protein pantothenic acidification levels ( Figure 6 F, G); Regarding histone modifications, PE treatment significantly upregulated H3K18 lactation, while GBP5 knockdown further enhanced this modification (F, G). Figure 6In contrast, GBP5 overexpression significantly inhibited PE-induced H3K18 acetylation (Fig. 1H, Fig. 1J). Figure 6 I, K). In summary, the results showed that GBP5 played a negative regulatory role in PE-induced enhancement of myocardial glycolysis and lactic acid modification.

[0109] From the above results, it can be seen that in the myocardial hypertrophy disease model caused by coarctation of the aortic arch, GBP5 gene deficiency significantly promotes myocardial hypertrophy, fibrosis, and worsens cardiac function, and GBP5 gene overexpression significantly inhibits myocardial hypertrophy, fibrosis, and protects cardiac function. Therefore, the GBP5 gene has the effects of protecting cardiac function and inhibiting myocardial hypertrophy and fibrosis, and in particular, the GBP5 gene can inhibit the occurrence of myocardial hypertrophy-related diseases caused by coarctation of the aortic arch, and GBP5 plays a negative regulatory role in PE-induced enhancement of myocardial glycolysis and lactic acid modification.

[0110] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. Application of Guanylate Binding Protein 5 (GBP5) in preparation of a drug for preventing, alleviating or treating myocardial hypertrophy and fibrosis.

2. Use according to claim 1, wherein The amino acid sequence of the guanylate binding protein 5 is shown as SEQ ID NO.

1.

3. The use according to claim 1, wherein The myocardial hypertrophy is pathological myocardial hypertrophy.

4. Application of a coding sequence of guanylate binding protein 5 in preparation of a drug for preventing, alleviating or treating myocardial hypertrophy and fibrosis.

5. The use according to claim 4, wherein the compound is ###0002### The coding sequence of the guanylate binding protein 5 is shown as SEQ ID NO.

2.

6. The use according to claim 4, wherein the compound is ###0002### The myocardial hypertrophy is pathological myocardial hypertrophy.

7. Application of a vector containing the coding sequence of guanylate binding protein 5 according to claim 4 or 5 in preparation of a drug for preventing, alleviating or treating myocardial hypertrophy and fibrosis.

8. Use according to claim 7, wherein the compound is ###0002### The vector is an adenovirus expression vector overexpressing guanylate binding protein 5.

9. The use according to claim 7, wherein the compound is ###00003### 8 or ###00004### 9. The myocardial hypertrophy is pathological myocardial hypertrophy.

Images