CN111184736A - Application of inhibiting HMBOX1 gene expression - Google Patents
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- CN111184736A CN111184736A CN201910268414.5A CN201910268414A CN111184736A CN 111184736 A CN111184736 A CN 111184736A CN 201910268414 A CN201910268414 A CN 201910268414A CN 111184736 A CN111184736 A CN 111184736A
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Abstract
The invention relates to application of inhibiting HMBOX1 gene expression in preparation of a medicine for treating pathological cardiac remodeling. According to the invention, the expression of HMBOX1 in swimming tissues is reduced, and the expression in myocardial ischemia-reperfusion tissues is abnormally increased through a protein immunoblotting method, and further functional experiments show that the down-regulated HMBOX1 plays a role in protecting pathological cardiac remodeling after myocardial ischemia-reperfusion. Accordingly, the HMBOX1 can be applied to the diagnosis of pathological cardiac remodeling or the development of drugs for inhibiting pathological cardiac remodeling.
Description
Technical Field
The invention relates to application of inhibiting HMBOX1 gene expression in preparation of a medicine for treating pathological cardiac remodeling.
Background
Pathological cardiac remodeling is an adaptive change after the heart is subjected to pathological stimuli such as ischemia, hypoxia, load increase and the like, and is an important stage for the development of many cardiovascular diseases, which can lead to the reduction of cardiac function, the fibrosis of the heart and finally the development of heart failure. Clinically, the most critical and effective method for acute myocardial infarction patients is reperfusion therapy in early ischemia, which effectively improves the mortality rate of acute myocardial infarction, but the process of recovering blood flow after myocardial tissue ischemia also causes a great deal of myocardial cell necrosis and apoptosis, and the phenomenon is called myocardial ischemia reperfusion injury. Clinically, common pathological cardiac remodeling can occur in the remodeling stage after myocardial ischemia reperfusion injury, and can also occur in other cardiovascular diseases such as hypertension, valve regurgitation, myocardial infarction and the like.
Exercise training is an adjuvant therapy for the prevention and treatment of various cardiovascular diseases. Unlike pathological cardiac remodeling, physiological cardiac remodeling promotes increased cardiomyocyte volume and number without loss of cardiomyocytes, cardiac fibrosis, and decreased cardiac function. Relevant studies at home and abroad show that lasting regular movement can promote physiological hypertrophy and cardiac regeneration of the heart, thereby protecting pathological cardiac remodeling caused by myocardial ischemia-reperfusion injury and aortic arch constriction. In conclusion, finding the diagnosis index and the intervention target of pathological cardiac remodeling from the molecular level has important clinical significance.
Transcription factors are a large group of protein molecules which can be specifically combined with a specific sequence at the upstream of the 5' end of a gene so as to ensure that a target gene is expressed at a specific strength in a specific time and space. There are three possible modes of action: 1) binding to the regulatory site of the promoter to prevent binding of other transcription factors; 2) acts on other transcription factors and inhibits the action of other factors; 3) transcription is prevented from occurring by altering the higher order structure of the DNA. The target gene may be covalently bound to the DNA binding domain of the transcription factor, thereby inhibiting or enhancing the expression of the gene. Previous studies have shown that transcription factors are involved in a wide range of physiological processes including development, immunity, and the body's response to stress. Analysis of transcription factor expression, activity, regulation, and gene sequence helps researchers determine the significance of their role in physiological and pathological processes. In 2011C, facial and the like (methods and compositions for treating or preventing pathological cardiac remodeling and heart failure, publication No. CN102099030A) administered PDE1 inhibitors to patients on the premise of effectively preventing and treating pathological cardiac remodeling and heart failure caused thereby. In addition, no other inhibitor is invented for diagnosing and treating pathological cardiac remodeling.
Disclosure of Invention
One of the purposes of the invention is to provide an application of inhibiting the expression of HMBOX1 gene in the preparation of drugs for treating pathological cardiac remodeling.
The pathological cardiac remodeling is as follows: pathologic cardiac remodeling caused by myocardial ischemia reperfusion injury, hypertension, valve regurgitation and myocardial infarction and other cardiovascular diseases.
In the invention, the HMBOX1 is a transcription factor with a regulation effect on pathological cardiac remodeling, and the HMBOX1 is obtained by first cloning in human pancreatic cDNA, but the function of the HMBOX1 is rarely researched so far. HMBOX1 has been found to localize in the nucleus as a transcription factor inhibiting gene expression, and to act as a telomere binding protein regulating cellular senescence. In addition to being localized to the nucleus, HMBOX1 was also found to be localized in the cytoplasm of vascular endothelial cells, inhibiting apoptosis and promoting autophagy through interaction with MT 2A.
The gene expression for inhibiting the HMBOX1 gene in the invention is a conservative nucleotide sequence, and the nucleotide sequence is as follows:
Forward：ccgggcctagctgtcatggaaagttctcgagaactttccatgacagctaggctttttg
Reverse：aattcaaaaagcctagctgtcatggaaagttctcgagaactttccatgacagctaggc
preparing a targeted drug for treating pathological cardiac remodeling, wherein the targeted drug is characterized in that: the main component of the drug is an inhibitor of HMBOX1, and the target of the inhibitor is not limited to HMBOX1 itself, but also comprises downstream molecules of HMBOX1, such as ETS1 and the like. The source of the drug comprises natural or artificial synthesis, or is obtained by transfecting cells with a vector capable of over-expressing the drug; the inhibitor can inhibit the expression of HMBOX1 in cells and tissues, or can disrupt the stability of HMBOX1 in cells and tissues, or can reduce the activity of HMBOX1 in cells and tissues, or can reduce the effective duration of action of HMBOX1 in cells or tissues; the inhibitor may be selected from: proteins, small molecule compounds, oligonucleotide expression vectors; the pharmaceutical carrier includes but is not limited to diluent, buffer, suspension, emulsion, granule, encapsulating agent, excipient or adsorption carrier, and the final therapeutic effect is that the expression amount of HMBOX1 in cells or tissues can be inhibited.
The research finds that the expression of HMBOX1 is reduced in heart tissues swimming in mice and abnormally increased in myocardial ischemia-reperfusion tissues, and further functional experiments show that the down-regulation of HMBOX1 can resist pathological heart reconstruction after myocardial ischemia-reperfusion injury, and ETS1 is a downstream molecule of the effect of the down-regulation. In 2009, zhangjian et al (recombinant human HMBOX1 expression vector, its expression product and antibody and application, publication No. CN101555484) discovered recombinant human HMBOX1 expression vector, its expression product, antibody and application. In 2009, senecio scandens et al (application of human transcription factor HMBOX1 gene, publication No. CN101555486) found application of HMBOX1 gene to natural killer cell regulation after overexpression. Based on the innovative discovery of the HMBOX1, diagnostic tools and targeted drugs related to the HMBOX1 for pathological cardiac remodeling are to be developed.
According to the invention, the expression of HMBOX1 in swimming tissues is reduced, and the expression in myocardial ischemia-reperfusion tissues is abnormally increased through a protein immunoblotting method, and further functional experiments show that the down-regulated HMBOX1 plays a role in protecting pathological cardiac remodeling after myocardial ischemia-reperfusion. Accordingly, the HMBOX1 can be applied to the diagnosis of pathological cardiac remodeling or the development of drugs for inhibiting pathological cardiac remodeling.
Drawings
Fig. 1 shows that HMBOX1 is down-regulated in the heart after swimming training and up-regulated in myocardial infarct zone 3 weeks after myocardial ischemia reperfusion injury. (A) C57BL/6 adult male mice were subjected to 3-week swimming training and immunoblotting showed that HIPK1 expression was down-regulated in swimming heart tissue (n-5: 6). (B) Adult mice were selected and subjected to coronary artery left anterior descending ligation, loosened after 30 minutes, and heart tissue samples were collected 3 weeks after open ligation. The immunoblotting method shows that HIPK1 is up-regulated in myocardial infarction area of myocardial ischemia-reperfusion injury (n-3). P < 0.05; p < 0.01.
Fig. 2 shows that inhibition of HMBOX1 promotes cardiomyocyte hypertrophy in neonatal rats, but does not affect cardiomyocyte proliferation, (a) EdU and cardiomyocyte-specific marker gene α -actin two-color immunofluorescence staining, (B) knockdown of HMBOX1 does not affect cardiomyocyte proliferation (n-4), (C) knockdown of HMBOX1 promotes cardiomyocyte hypertrophy (n-4), P < 0.01.
Fig. 3 is a graph showing that inhibition of HMBOX1 could counteract the induction of neonatal rat cardiomyocyte apoptosis by Oxygen Glucose Deprivation Recovery (OGDR) (a) Tunel and two-color immunofluorescent staining of the cardiomyocyte-specific marker gene α -actin. the knock-down of HMBOX1 at a scale of 100 μm (B) reduced the OGDR-induced apoptosis rate of neonatal rat cardiomyocytes (n 4), P < 0.05;, P < 0.01.
Fig. 4 shows that HMBOX1 is expressed in cardiomyocytes in an enriched manner. (A) qPCR identified marker gene expression (n-4-6) of sorted primary cardiomyocytes (NRCM) and fibroblasts (NRCF). (B) qPCR showed that the expression level of HMBOX1 was significantly higher in cardiomyocytes than in fibroblasts (n-4-6). (C) The bioinformatics Software (genomatic Software Suite) screened ETS1 as a downstream molecule of HMBOX 1. P < 0.01; p < 0.001.
Fig. 5 shows that inhibition of HMBOX1 protects against pathological cardiac remodeling caused by myocardial ischemia-reperfusion injury. (A) Protein immunoblotting confirmed the efficiency of knock-down of the HMBOX1 virus in myocardial tissue (n-3). (B) C57BL/6 adult male mice are selected to be subjected to coronary artery Left Anterior Descending (LAD) ligation, the ligation is loosened after 30 minutes, left ventricular ejection fraction (EF,%) and left ventricular short axis shortening rate (FS,%) are detected by using cardiac ultrasound of the small animals 3 weeks after the open ligation, and the heart function of the mice can be remarkably improved by inhibiting HMBOX1 (n is 6-7). P < 0.05; p < 0.01.
Detailed Description
Referring to fig. 1-5, the technical solution of the present invention is further described below with reference to specific embodiments. It is to be understood that the following examples are illustrative of the invention and are not to be construed as limiting the invention in any way.
1. Isolation and culture of Neonatal Rat Cardiomyocytes (NRCM) and fibroblasts (NRCF): 27.2g NaCl, 19.04g HEPES, 0.552g NaH2PO4, 2.4g Glucose, 1.6g KCl, 0.82g MgSO4 & 7H2O were weighed to adjust the pH of the solution to 7.4 and sterilized ddH 2O-400 mL were added to make up 10 × ADS for use. Weighing collagenase powder 0.016g and pancreatin powder 0.024g, preparing pancreatin collagenase mixture 40mL with 1xADS, and filtering with 0.22 μm filter head. Sterilizing the chest and abdomen of a rat just born with 75% ethanol, taking the heart, gently squeezing the blood in the heart with forceps, fully shearing the heart in a glass bottle, adding a proper amount of pancreatin collagenase mixed liquor to fully mix with the heart tissue blocks, then placing the glass bottle into a shaking table (set at 37 ℃ in advance) to shake and digest at 120rpm, collecting the digestive juice into a centrifuge tube after 20min, and adding horse serum to stop digestion. Then the mixed solution is centrifuged at 1200rpm for 5min, the supernatant is discarded, the mixed solution is resuspended by using a culture medium, the steps are repeated until the tissue block is fully digested, the cell suspension is filtered into a culture dish by using a filter screen, and the fibroblast is obtained after differential adherence for 60 min (the fibroblast adheres faster than the myocardial cell). Nonadherent cells are collected and subjected to density gradient centrifugation to obtain cardiomyocytes. And (4) plating according to the number of cells required by the experiment, culturing by using a myocardial cell culture medium, and carrying out the experiment after the cells are spread.
2. Immunofluorescence staining (EDU and α -actin co-staining) removing the culture medium, washing with PBS 3 times, 5min each time, fixing the cells at room temperature for 30min (4% PFA), washing with PBS 3 times, 5min each time, breaking the membrane for 20min (0.5% TritonX-100), washing with PBS 3 times, 5min each time, sealing at room temperature for 2h (5% BSA), incubating the primary antibody at 4 ℃ for 12h (α -actin: 5% BSA: 1: 200), discarding the primary antibody the next day, washing with PBS 3 times, 5min each time, incubating the secondary antibody at room temperature in the dark for 2h (secondary antibody: 5% BSA: 1: 200), washing with PBS 3 times, 5min each time, staining EDU in the dark according to Ky-base EDU, staining with DAPI for 20min cell nucleus PBS 3 times, 5min each time, and taking a picture of the sample.
3. The Oxygen Glucose Deprivation Recovery (OGDR) model induces cardiomyocyte apoptosis: after the control group is transfected with the sh-Scramble and sh-HMBOX1 viruses, the whole process is normally cultured, after the experimental group is transfected with the sh-Scramble and sh-HMBOX1 viruses for 36 hours, the culture medium of the cells is changed into a sugar-free culture medium, the cell culture plate is placed in an oxygen-deficient box to be incubated for 8 hours, the cell culture plate is taken out from the oxygen-deficient box to be subjected to reoxygenation operation, the sugar-free culture medium is changed back to the culture medium of normal cardiac muscle cells, and the cells are collected after being incubated for 12 hours to be subjected to Tunel staining.
TUNEL staining (TUNEL staining and α -actin co-staining) by removing the medium, washing with PBS 3 times for 5min, adding 100. mu.l of 4% paraformaldehyde to each well, fixing at room temperature for 30min, washing with PBS 3 times for 5min, adding 100. mu.l of glycine to each well, washing with PBS 5min, adding 100. mu.l of 0.5% Triton to each well, disrupting membranes for 20min, washing with PBS 3 times for 5min, adding 100. mu.l of 0.01M PBS to each well, blocking with 5% BSA prepared by 5% BSA: α -actin ═ 200:1, incubating in refrigerator at 100. mu.l for 4 ℃ overnight, removing one antibody the second day, washing for 3 times for 5min, incubating in PBS 5min, adding 5% BSA: 200:1, incubating in PBS for 2h, incubating at room temperature for 3 times, incubating in PBS 5min, adding PBS for 3 min, incubating in PBS for 5min, adding 5min, diluting with 5% BSA, washing with PI 5 μ g Buffer solution for 5, and diluting with 5 μ g Buffer for 5 g wash at room temperature, and adding PI 5 μ g wash for 5 g wash, and water for 5min, and adding 5 μ g wash for 5 g wash.
5. Western blot detection of HMBOX 1: cell and tissue proteins were lysed by adding 1% PMSF to RIPA lysate and total Protein concentration was determined by using BCA Protein Assay Kit. Mixing the protein supernatant with 5 × Loading buffer (4:1), water-bathing at 100 deg.C for 5min, cooling, and storing at-20 deg.C. SDS gel of the desired concentration was prepared, and an equal amount of total protein was loaded, subjected to gel electrophoresis, and transferred to a PVDF membrane. Sealing with 5% skimmed milk powder for 2 hr. Primary antibody HMBOX1 was prepared from 5% nonfat dry milk at a ratio of 1:1000 and incubated overnight at 4 ℃ with shaking. The next day, washing with TBST (3 times for 10min each time), preparing corresponding secondary antibody with 5% skimmed milk powder at a ratio of 1:10000, and incubating at room temperature for 2 hr. Washing with TBST, final exposure to light and development using ECL luminescence kit in a color developing instrument, and measurement of gray scale values of protein bands using Image J software using GAPDH as an internal reference.
6. Extracting cell RNA and detecting HMBOX1, cTnT, cTnl, Col3a1 and Col1a 1: total RNA was extracted from cells using RNeasy MiniKit (Qiagen). Relative detection of HMBOX1, cTnT, cTnl, Col3a1 and Col1a1 by SYBR methodExpression level, 18S as reference primer, 2-ΔΔCtAnd (4) carrying out statistics by the method.
7. Mouse myocardial ischemia reperfusion injury model (IRI) induced pathologic cardiac remodeling model: firstly, anesthetizing the mouse by injecting the anesthetic into the abdominal cavity, fixing the four limbs of the mouse on a heat blanket by using a medical adhesive tape after the mouse is completely anesthetized, and depilating the hair by using the depilatory cream. Then disinfecting the operation area by using 75% alcohol, carrying out microshearing to cut the neck skin of the mouse, separating the neck muscle and tissue in a blunt manner, exposing the neck muscle and tissue out of the trachea, connecting the neck muscle and tissue with a breathing machine, and starting the operation after observing that the breathing frequency of the mouse is the same as the breathing frequency of the breathing machine. Cutting an opening at the fourth intercostal and the fifth intercostal of the left chest of the mouse by using small scissors under a scope by means of a stereoscope, exposing the heart, slightly separating the pericardium, finding the position of the left anterior descending branch of the heart, performing ligation by using a suture line with a thread, and observing that the area below a ligation site becomes white after 30s, which indicates that the ligation is successful, and starting timing. The left anterior descending artery is ligated for 30min before ischemia, then the ligature is cut off, then the rib is sutured, the muscle is sutured, finally the skin is sutured. Observing the respiration condition of the mouse, pulling out the trachea cannula after the mouse recovers the spontaneous respiration, putting the mouse back to the mouse cage for feeding, and measuring the cardiac function of the mouse by using cardiac ultrasonic of the mouse 3 weeks after the operation.
8. Ultrasonic measurement of mouse cardiac function in small animal hearts: depilatory cream is used for depilating the chest of a mouse, the mouse is anesthetized by isoflurane inhalation anesthesia, four limbs of the mouse are fixed, a proper amount of chelating agent is smeared on the heart of the mouse, the heart of the mouse is found by a probe, the long axis of the left ventricle and the short axis of the left ventricle beside the sternum are taken for examination after the heart rate is stable, the cardiac function is measured and calculated, and the left ventricular ejection fraction EF and the left ventricular short axis shortening rate FS are obtained.
Sequence listing
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Claims (2)
1. An application of suppressing HMBOX1 gene expression in preparing the medicines for treating pathological cardiac remodeling is disclosed.
2. The use according to claim 1, wherein said pathological cardiac remodeling is: pathologic cardiac remodeling caused by ischemia-reperfusion injury, hypertension, valve regurgitation and myocardial infarction.
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Non-Patent Citations (3)
| Title |
|---|
| XIAOJUN LIU ET AL.: "miR-222 is necessary for exercise-induced cardiac growth and protects against pathological cardiac remodeling", 《CELL METAB》 * |
| 姚建华等: "运动诱导生理性心肌肥厚的分子机制", 《中华心力衰竭和心肌病杂志》 * |
| 罗婠等: "mir-222在急性心肌梗死诊断的意义", 《中国实验诊断学》 * |
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Application publication date: 20200522 |