CN116270993A - Medical application of Trim55 protein in preventing or treating heart failure after myocardial infarction - Google Patents
Medical application of Trim55 protein in preventing or treating heart failure after myocardial infarction Download PDFInfo
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- CN116270993A CN116270993A CN202310290871.0A CN202310290871A CN116270993A CN 116270993 A CN116270993 A CN 116270993A CN 202310290871 A CN202310290871 A CN 202310290871A CN 116270993 A CN116270993 A CN 116270993A
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Abstract
The invention discloses an application of a Trim55 (Trim 55), in particular relates to an application of Trim55 protein or an active fragment thereof in preparation of drugs for preventing and/or treating myocardial apoptosis and heart failure after myocardial infarction, and belongs to the field of biomedical application. By studying animals and cells, the molecular mechanism of Trim55 protein or active fragments thereof in myocardial apoptosis and heart failure after myocardial infarction is disclosed. Also relates to the use of recombinant vectors or recombinant cells expressing Trim55 protein or an active fragment thereof in the preparation of a medicament for preventing and/or treating myocardial apoptosis and heart failure after myocardial infarction. The invention provides a new target for diagnosis and treatment of myocardial apoptosis and heart failure after myocardial infarction, and has important clinical significance.
Description
Technical Field
The invention belongs to the field of medicines, relates to a medical application of Trim55 (Tripartite motif containing, trim 55) containing a three-way motif, and in particular relates to a medical application of Trim55 protein in preventing or treating heart failure after myocardial infarction.
Background
Cardiovascular disease is one of the important diseases that seriously jeopardizes human health. The Chinese cardiovascular health and disease research report 2021 indicates that the prevalence of cardiovascular diseases in China is in a continuously rising stage, and cardiovascular disease death is the first leading cause of total death of urban and rural residents, wherein coronary atherosclerotic heart disease (coronary heart disease) is the disease with the highest prevalence. Myocardial Infarction (MI) is the most severe disease type in coronary heart disease, and MI mortality generally has a rapid rise since 2005. MI has a high incidence and mortality worldwide, severely threatening the health of humans. While percutaneous coronary intervention and thrombolytic therapy can save the life of the patient, clinical trials have shown that patients have poor long-term prognosis, most of which are Heart Failure (HF). Studies have shown that activation of inflammation, cardiomyocyte death and fibroblast proliferation can lead to myocardial fibrosis, thereby exacerbating the occurrence of post-MI HF, however, the key regulatory molecules and key mechanisms of post-MI HF are currently unknown, and there is a lack of effective targeted therapeutic drugs. Therefore, the occurrence mechanism of HF after MI is defined, and the search of a new control target point has important social significance and clinical significance.
In early stages after MI, the number of myocardial cells is reduced due to ischemia necrosis, and other myocardial cells undergo apoptosis under the action of injury factors such as oxidative stress and inflammatory reaction, so that myocardial cells are further reduced, and cardiac function is reduced to generate HF. Apoptosis is a central event in post-MI pathological processes. After MI, myocardial cells are ischemic and hypoxic, death receptors bind to ligands or mitochondria release cytochrome C to initiate apoptosis, causing activation of Caspase (Caspase) family 8 or 9, thereby causing a cascade of reactions, ultimately Caspase3 is activated, and an apoptosis program is performed. Studies have shown that the absence of Bax (a member of the Bcl-2 family that induces apoptosis) reduces infarct size and cardiac remodeling during MI. Thus, by elucidating the molecular mechanisms of myocardial apoptosis in cardiac remodeling, targeted therapeutic intervention may be an important research direction to improve post-myocardial infarction outcome.
Trim55 (Trim 55), an E3 ubiquitin ligase specific for heart and skeletal muscle, also known as MURF2 or RNF29, is a member of the Trims family, consisting of a zinc binding domain, a ring finger domain, a B box motif and a coiled coil domain. Trim55 is involved in early myofibrillar formation in physiological conditions, and its loop refers to the interaction of the B-box domain with the transactivation domain of Serum Response Factor (SRF) to inhibit SRF activity and nuclear localization, and to participate in and influence muscle cell transcription; as a main member in the development process of mouse heart embryo, plays a role in dynamic regulation. Previous studies have shown that Trim55 knockout mice develop severe hypertrophy and premature contractile dysfunction in a diabetic cardiomyopathy model, and that Trim55 inhibits cardiac hypertrophy after diabetes by ubiquitination of cardiac pparα and pparγ1 regulation; mice lacking Trim63 and Trim55 developed myocardial and skeletal muscle hypertrophy, but Trim55 knockout mice had no apparent myocardial hypertrophy in the aortic stenosis (TAC) induced myocardial hypertrophy model. Trim55 expression was significantly increased in chronic heart failure models and myocardial ischemia reperfusion models, and administration of overexpressed Trim55 after myocardial ischemia reperfusion could further promote myocardial apoptosis. However, the role and mechanism of Trim55 after MI is not yet clear. The role of Trim55 in MI and its mechanism are therefore worthy of intensive investigation.
Disclosure of Invention
In view of the defects existing in the prior art, the inventor of the invention discovers through a large number of experiments that after the MI of a C57BL/6J mouse, the mRNA and protein expression of the Trim55 gene gradually rise along with the time gradient; the left ventricular contractile function of mice with Trim55 knocked out systemically after MI is significantly improved, and myocardial apoptosis and myocardial fibrosis are reduced. post-MI left ventricular contractile function of mice over-expressing Trim55 systemically is significantly reducedDecrease, myocardial apoptosis and myocardial fibrosis increase. Cytological studies have found that cobalt chloride (CoCl 2 ) Trim55 gene mRNA and protein expression were significantly increased following stimulation, and apoptosis index (clear caspase 3) protein expression was increased. Overexpression of Trim55 will cause CoCl 2 The expression of the induced myocardial apoptosis protein is further increased, while the low expression of Trim55 reduces CoCl 2 Induced cardiomyocyte apoptosis. The results indicate that Trim55 protein can be used to prevent or treat HF caused by abnormal ventricular remodeling due to increased apoptosis after MI. The present invention has been completed based on the above findings.
In order to achieve the above object, the present invention adopts the following technical scheme.
The invention provides an application of Trim55 protein or an active fragment thereof in preparing a medicament, which is characterized in that the medicament is used for preventing and/or treating heart failure caused by abnormal heart reconstruction caused by excessive apoptosis of myocardial cells after MI.
Further, the pharmaceutical composition comprises an inhibitor of Trim55 protein or an active fragment thereof.
The invention also provides the use of a nucleic acid molecule encoding Trim55 protein or an active fragment thereof, a recombinant vector or a recombinant cell expressing Trim55 protein or an active fragment thereof in the preparation of a medicament, characterized in that the medicament is used for preventing and/or treating heart failure caused by abnormal heart remodeling due to excessive apoptosis of myocardial cells after MI.
Further, the pharmaceutical composition comprises a nucleic acid molecule encoding a Trim55 protein or an active fragment thereof, a recombinant vector expressing a Trim55 protein or an active fragment thereof, or an inhibitor of a recombinant cell.
The invention also provides the use of an agent capable of inhibiting up-regulation of Trim55 protein or an active fragment thereof or promoting down-regulation of Trim55 protein or an active fragment thereof in the manufacture of a medicament for the prevention and/or treatment of heart failure caused by abnormal heart remodeling due to excessive apoptosis of cardiomyocytes after MI.
The invention also provides a pharmaceutical composition for preventing and/or treating heart failure caused by abnormal heart remodeling caused by excessive apoptosis of cardiac myocytes after MI, which is characterized by comprising one or more of the following active ingredients:
(1) An agent capable of inhibiting upregulation of Trim55 protein or active fragment thereof expression;
(2) An agent that promotes down-regulation of Trim55 protein or an active fragment thereof;
(3) A nucleic acid molecule encoding a Trim55 protein or an active fragment thereof, a recombinant vector or a recombinant cell expressing a Trim55 protein or an active fragment thereof.
Further, the composition further comprises a pharmaceutically acceptable carrier or excipient; the dosage form of the pharmaceutical composition includes a pharmaceutically acceptable dosage form.
The invention also provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition contains Trim55 protein or an active fragment thereof, a nucleic acid molecule encoding Trim55 protein or an active fragment thereof, a recombinant vector or a recombinant cell expressing Trim55 protein or an active fragment thereof, or an agent capable of inhibiting down-regulation of Trim55 protein or an active fragment thereof, and optionally a pharmaceutically acceptable carrier or excipient; the composition is used for preventing and/or treating heart failure caused by abnormal heart remodeling caused by excessive apoptosis of cardiac muscle cells after MI.
The invention also provides application of the reagent for detecting the expression level of the Trim55 protein or the active fragment thereof in preparing a kit for predicting and/or evaluating the treatment effect and prognosis of myocardial cell excessive apoptosis after MI.
The invention also provides a kit for detecting excessive apoptosis of myocardial cells after MI, which is characterized by comprising a reagent for detecting the expression level of Trim55 protein or an active fragment thereof.
In the present invention, the mammal may be, for example, a rat, a mouse, a dog, a mini pig, a monkey, a human, or the like.
In the invention, the active fragment of the Trim55 protein refers to a fragment with Trim55 protein function, which can be a part of Trim55 protein or a fragment obtained by deleting, adding or replacing an amino acid sequence of Trim55 protein; the site which possibly affects the activity is avoided as required by a person skilled in the art, and other sites are modified by deletion, addition or replacement, etc., so that the modified Trim55 protein still has the activity or the function of the Trim55 protein.
In the present invention, the post-MI myocardial injury has a meaning well known in the art, and refers to left ventricular dysfunction occurring after MI.
In the present invention, the prevention and/or treatment of excessive apoptosis of myocardial cells after MI refers to inhibition or alleviation of elevation of apoptotic proteins.
In the present invention, the use of the expression level of Trim55 protein or an active fragment thereof for prediction and/or evaluation means that when the expression level of Trim55 protein or an active fragment thereof in blood, tissue or cells is lower than a reference value, it is possible to predict myocardial damage and excessive apoptosis of myocardial cells after MI, or evaluate the therapeutic effect or prognosis thereof.
In the present invention, the expression level of Trim55 protein or an active fragment thereof can be detected by a method well known in the art, for example, by amplifying Trim55 mRNA by polymerase chain reaction and performing a quantitative reaction, or by detecting Trim55 protein expression level using Western Blot.
In the present invention, the expression level of the protein refers to the level of mRNA or the level of the protein.
In the present invention, the up/down regulation of protein expression in a tissue/cell means increasing or decreasing the protein level or mRNA level in a tissue/cell by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by more than 100%. Wherein said up-or down-regulation is compared to non-intervening tissues or cells.
In the present invention, the agent capable of inhibiting down-regulation of Trim55 protein or an active fragment thereof or promoting up-regulation of Trim55 protein or an active fragment thereof is known in the art.
Compared with the prior art, the invention has the following advantages and beneficial effects.
The inventor of the invention firstly proposes C57BL through a plurality of experimentsAfter MI of 6J mice, trim55 gene mRNA and protein expression gradually increased with time gradient; the left ventricular contractile function of mice with Trim55 knocked out systemically after MI is significantly improved, and myocardial apoptosis and myocardial fibrosis are reduced. Left ventricular contractile function was significantly reduced after MI in mice overexpressing Trim55 throughout the body, and myocardial apoptosis and myocardial fibrosis were increased. Cytological studies have found that cobalt chloride (CoCl 2 ) Trim55 gene mRNA and protein expression were significantly increased following stimulation, and apoptosis index (clear caspase 3) protein expression was increased. Overexpression of Trim55 will cause CoCl 2 The expression of the induced myocardial apoptosis protein is further increased, while the low expression of Trim55 reduces CoCl 2 Induced cardiomyocyte apoptosis. The results show that the Trim55 protein can be used for preventing or treating abnormal ventricular remodeling caused by the aggravation of apoptosis after MI, provides a new target for diagnosis and treatment of myocardial apoptosis and heart failure after myocardial infarction, and has important clinical significance.
Drawings
FIG. 1 shows that MI border region Trim55 gene transcription and protein expression are significantly increased after myocardial infarction. Wherein A-B is Western blot for detecting Trim55 protein expression in heart infarction marginal areas of MI mice at different time points; c is fluorescence quantitative PCR detection of Trim55 protein expression in the myocardial infarction marginal area of the MI mice; d is immunohistochemical staining to detect Trim55 protein expression in the heart infarction border area of the MI mice at different time points; e is Western blot detection of CoCl 2 After stimulation of rat cardiomyocytes (H9C 2), the apoptosis protein clear caspase3 and Trim55 expression; f is fluorescence quantitative PCR detection CoCl 2 Trim55 mRNA expression after stimulation of rat cardiomyocytes (H9C 2).
FIG. 2A full body knockout Trim55 mouse (Trim 55) -/- ) Is established and identified. Wherein A is a gene knockout mouse establishment scheme and PCR identification; B-C is Western blot to detect the knockout efficiency of the gene knockout mouse; d is the knock-out efficiency of the fluorescent quantitative PCR detection gene knockout mouse.
FIG. 3 Whole body knockout Trim55 mice (Trim 55) -/- ) Reduce myocardial injury after MI. Wherein A is a survival curve; b is the ratio of heart weight to body weight; c is the ultrasonic evaluation of the left ventricle of the small animalShrink functions EF%, FS%, and LVID; d is HE and Masson dyeing to detect the size of the infarct area and the fibrosis condition of the infarct edge area; E-F is protein expression of fibrosis indexes Collagen I and TGF-beta of a heart infarction border region detected by Western blot.
FIG. 4 Whole body knockout Trim55 mice (Trim 55) -/- ) Reduce myocardial cell apoptosis in the infarct border area after MI. Wherein A-B is Western blot detection of apoptosis index of the edge region of heart infarction; c is TUNEL staining to detect myocardial apoptosis in infarct border zone.
FIG. 5A mice with systemic over-expression of Trim55 (AAV-Trim 55) exacerbate post-MI myocardial injury. Wherein A is the over-expression efficiency of a Western blot detection over-expression Trim55 mouse; b is the over-expression efficiency of the fluorescent quantitative PCR detection over-expression Trim55 mouse; c is a survival curve; d is the ratio of heart weight to body weight; e is a small animal ultrasound evaluation of left ventricular contractile function EF%, FS% and LVAW; f is HE and Masson dyeing to detect the size of the infarct area and the fibrosis condition of the infarct edge area; G-H is protein expression of fibrosis indexes Collagen I and TGF-beta of a heart infarction border region detected by Western blot.
FIG. 6. Systemic over-expression Trim55 (AAV-Trim 55) mice exacerbate post-MI infarct border zone myocardial apoptosis. Wherein A-B is apoptosis index of Western blot detection heart infarction marginal area; c is TUNEL staining to detect myocardial apoptosis in infarct border zone.
Fig. 7. Promotion of hypoxia-induced apoptosis of cardiomyocytes following cytologic administration of overexpressed Trim 55. Wherein A-B is apoptosis index after Western blot detects myocardial cells over-express Trim 55; C-D is Western blot to detect myocardial cell over-expression Trim55 and CoCl is given 2 A post apoptosis indicator; e is flow cytometry to detect cardiomyocyte over-expression Trim55 and to administer CoCl 2 Apoptosis after; f TUNEL staining to detect cardiomyocyte over-expression Trim55 and CoCl administration 2 Post-apoptotic conditions.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The experimental data of the present invention are all percentages. The comparison of the two sample rates uses chi-square test and the statistical processing uses Graphpad prism8.0 software package processing. p <0.05 is statistically different.
Example 1: after the establishment of the MI model of the C57BL/6J mice, trim55 expression was up-regulated with time gradient.
(1) Establishment of MI model of C57BL/6J mice.
Firstly, the mice are inhaled and anesthetized by isoflurane, the skin is gently sheared between 3 rd and 4 th ribs of the left chest of the mice by ophthalmic scissors, after a small opening is formed in the pleura of the mice in a blunt way, the heart is rapidly extruded by the left hand and the right hand, the left anterior descending branch of the coronary artery of the heart is found, the heart is rapidly ligated by using a 6-0 suture with a needle, the apex of the heart is observed to be whitened, the typical myocardial infarction of an electrocardiogram shows that the ligation is successful, the heart is then put back into the chest, the pneumothorax is rapidly prevented from dying by action, and finally the skin is sutured layer by layer. Ultrasound examination was performed 3 days and 28 days after surgery, respectively, to determine whether the myocardial infarction model was successful.
(2) Western blot detects protein expression of Trim55 in the border region of MI mice with heart infarct at different time points.
In order to detect the expression condition of Trim55 in the myocardial infarction border area of MI mice at different time points, RIPA lysate is used for extracting sham group proteins and the protein concentration of the mouse infarction border area at different time points is measured by a BCA method. Trim55 protein expression was detected by Western Blot. The method comprises the following steps: after 30 μg of protein was boiled at 95℃for 5min, the sample was loaded after a short centrifugation, and run on SDS-PAGE with 10% separation gel, loading was run to the bottom of the gel as the electrophoresis termination time. Transferring the sample onto a cellulose membrane in a sandwich mode at 90V voltage for 2 hours; and (3) sealing for 1-2 hours at normal temperature in 5% nonfat milk powder, adding the primary antibody, and incubating overnight at 4 ℃. Trim55 antibody (1:1000, thermo company, USA) and GAPDH antibody (1:1000, CST company, USA) were used as primary antibodies, horseradish peroxidase-labeled goat anti-rabbit antibody (CST company, USA) was used as secondary antibodies, and after 3 times of TBS-T washing, development was performed by using ECL kit (GE company, USA) by luminescence.
The results show that: trim55 protein levels increased in a time-dependent manner within 28d after MI compared to sham group (n=3, * p<0.05， ** p<0.01 (fig. 1A-B).
(3) Fluorescent quantitative PCR detects mRNA expression of Trim55 in the myocardial infarction border region of MI mice.
Tissue RNA was extracted by Trizol method, cDNA was obtained by reverse transcription using TakaRa reverse transcription kit, and quantitative PCR was performed by SYBRGreen method.
The quantitative PCR primer sequences were as follows:
the results show that: trim55 gene mRNA expression levels were significantly elevated after MI compared to sham group (n=5, ** p<0.01， *** p<0.001 (fig. 1C).
(4) Immunohistochemical staining of Trim55 examined the expression of Trim55 in the infarct border zone of MI mice.
1) Paraffin sections were dewaxed in sequence in xylene i, xylene ii, 100%, 95%, 90% and 85% alcohol solutions, and finally rinsed with distilled water for rehydration.
2) And (3) placing paraffin sections into antigen retrieval liquid prepared according to a ratio of 1:50, boiling the paraffin sections in boiling water at 100 ℃ for 40min, and then placing the paraffin sections under normal temperature conditions for closed cooling to room temperature.
3) The sections were removed, the heart tissue was circled with an immunohistochemical pen and placed in a wet box for further staining. 100. Mu.L of reagent A was gently added dropwise to the specimen, allowed to incubate at room temperature for 10min, and then rinsed 3 times with PBS for 3min each.
4) 100 mu L of the reagent B solution is dripped on a specimen, the specimen is placed in a room temperature environment for incubation for 10min, and the reagent B is directly thrown away without washing the specimen.
5) mu.L of the primary antibody solution prepared by using PBS according to a ratio of 1:100 was added dropwise to the specimen, and the specimen was left at 4℃overnight. The next day, paraffin sections were fully rewarmed at room temperature for 30min, and then rinsed with PBS solution for about 3min each time.
6) Diluting the solution C with PBS at a concentration of 1:100, dripping 100 mu L of diluted solution onto a specimen, standing for 15min at room temperature, and washing 3 times with PBS for 3min each time.
7) 100. Mu.L of the solution of the reagent D was added dropwise to the specimen for incubation at room temperature for 15min, followed by rinsing with PBS solution 3 times for 3min each.
8) And then dripping DAB chromogenic liquid prepared according to the specification on a specimen for dyeing, observing the dyeing condition under a mirror at any time, immediately flushing with PBS solution after the color development is finished to stop the color development, and washing for 3 times for 3min each time.
9) Nuclear staining, same as HE staining. The results were then observed under a microscope, and then photographed and saved.
The results show that: trim55 expression increased significantly with time gradient compared to sham group (fig. 1D).
(5) Western blot detection of CoCl 2 Trim55 and the apoptosis protein clear caspase3 expression after stimulation of cardiomyocytes (H9C 2).
See example 1(2 for specific methods). Trim55 antibody (1:1000, U.S. Thermo Co.), clear caspase3 antibody (1:1000, U.S. CST Co.), and GAPDH antibody (1:1000, U.S. CST Co.) were used as primary antibodies.
The results show that: compared with the control group, coCl 2 (300. Mu.M) stimulation, trim55 protein and clear caspase3 protein expression were significantly up-regulated in H9C2 cells (n=3, * p<0.05 To prompt CoCl 2 Increased Trim55 expression can be induced while apoptosis is induced (fig. 1E).
(6) Fluorescent quantitative PCR detection of MI mice CoCl 2 mRNA expression of Trim55 in cardiomyocytes (H9C 2) was stimulated.
Tissue RNA was extracted by Trizol method, cDNA was obtained by reverse transcription using TakaRa reverse transcription kit, and quantitative PCR was performed by SYBRGreen method.
The quantitative PCR primer sequences were as follows:
the results show that: compared with the control group, coCl 2 (300 μm) stimulation, trim55 gene mRNA expression in H9C2 cells was significantly elevated (n=3, * p<0.05 (fig. 1F), suggesting that hypoxia stimulation may up-regulate Trim55 expression.
Example 2: trim55 knockout mice were established and identified.
(1) Trim55 Knockout (KO) mice were obtained by the Jiangsu Jiuyaokang biotechnology company (Jiangsu, china) using CRISPR/Cas9 technology. Cas9 mRNA and gRNAs are produced by in vitro transcription. The primer is adopted: forward: TCAATTCTCAGACTTGTAGGTCGG, reverse: TATGAGACTACTTCCTGTGAGGCC (fig. 2A).
(2) Western blot detection Trim55 -/- Trim55 expression in mouse hearts.
See in particular example 1.Trim55 antibody (1:1000, thermo Inc. USA) and GAPDH antibody (1:1000, CST Co., USA) were used as primary antibodies.
The results show that: trim55 compared to C57 mice -/- Trim55 protein expression was significantly reduced in the mouse heart (n=3, * p<0.05 (fig. 2B-C).
(3) Fluorescent quantitative PCR detection Trim55 -/- Trim55 expression in mouse hearts.
The results show that: trim55 compared to C57 mice -/- Trim55 mRNA expression was significantly reduced in the mouse heart (n=3, * p<0.05 (fig. 2D).
Example 3: whole body knockout Trim55 mice (Trim 55) -/- ) Reduce myocardial injury after MI.
(1) MI-28d, the mice survival curve was counted.
The results show that: trim55 compared to the respective sham group -/- Survival was significantly reduced in both the-MI and C57-MI groups, but Trim55 -/- -MI survivalThe rate was higher than C57-MI (n=10, 14, 10, 14, p= 0.4801) (fig. 3A).
(2) MI-28d, heart to body weight ratio was counted after the mice were killed.
After weighing the body weight, killing the mice, taking the hearts, and weighing after removing the redundant fat to obtain the heart weight.
The results show that: trim55 compared to the respective sham group -/- The weight to weight ratio of the centers of both the-MI group and the C57-MI group was significantly increased, but Trim55 -/- The core-to-weight ratio of MI is significantly lower than that of C57-MI (n=10, 8, 10, ** p<0.01， *** p<0.001 (fig. 3B), prompt Trim55 -/- Significantly reduce post-MI cardiac enlargement.
(3) Mice were evaluated for cardiac contractile function by ultrasound.
Mice were dehaired, anesthetized with isoflurane and examined for systolic function using a Vevo2100 small animal sonicator. After the couplant is smeared on the front chest, an ultrasonic probe is placed on the front chest to find the longitudinal axis of the heart, an image is acquired, and the shrinkage function indexes EF% and FS% are obtained through measurement and analysis.
The results show that: trim55 compared to the respective sham group -/- The weight to weight ratio of the centers of both the-MI group and the C57-MI group was significantly reduced, but Trim55 -/- EF% and FS% of MI are significantly higher than C57-MI, LVID is significantly lower than C57-MI (n=10, 8, 10, * p<0.05， ** p<0.01， *** p<0.001 (fig. 3C), prompt Trim55 -/- Can significantly improve the abnormal cardiac function of mice.
(4) HE staining detects myocardial infarction area and Masson staining detects the degree of myocardial fibrosis.
HE staining step.
1) Paraffin sections were dewaxed in sequence in xylene i, xylene ii, 100%, 95%, 90% and 85% alcohol solutions, and finally rinsed with distilled water for rehydration.
2) And (3) placing the dewaxed transparent paraffin section into a hematoxylin solution for dyeing for 8-10 min, and then placing the paraffin section into running water for flushing.
3) The paraffin sections treated as described above were placed in 1% hydrochloric acid alcohol for complete differentiation for 30s to 60s, and then placed in running water for sufficient washing.
4) And (3) placing the paraffin section processed in the steps in ammonia water for 1-2 min for blue returning, and then placing the paraffin section in running water for flushing.
5) The treated slices are fully immersed in the prepared water-soluble eosin solution for 10min, and then are fully washed under running water.
6) Paraffin sections were placed in 90%, 95%, 100% alcohol, xylene i and ii solutions in sequence for adequate transparency.
7) The clear paraffin sections were placed in a fume hood and sealed with neutral gum, taking care that the sheet could not be dried.
8) And (5) observing by a microscope.
The Masson staining kit was used, and the specific procedure was as follows.
1) Paraffin sections were dewaxed in sequence in xylene i, xylene ii, 100%, 95%, 90% and 85% alcohol solutions, and finally rinsed with distilled water for rehydration.
2) And (3) placing the dewaxed transparent paraffin section into a hematoxylin solution for dyeing for 8-10 min, and then placing the paraffin section into running water for flushing.
3) And (3) placing the processed paraffin sections in 1% hydrochloric acid alcohol for complete differentiation for 30s to 60s, reversing blue in ammonia water for 1-2 min, and then placing the paraffin sections in running water for full flushing.
4) Ponceau acid fuchsin liquid dyeing is carried out for 5-10 min, and washing is carried out for 1min by weak acid solution (or 1-3% glacial acetic acid solution).
5) And treating with 1% phosphomolybdic acid solution for about 1-2 min.
6) And (3) directly counterstaining with aniline blue solution for 2min after throwing away the phosphomolybdic acid solution without washing with water, and washing with weak acid solution for 1min.
7) Sequentially adding into 95% alcohol and 100% alcohol I and II, respectively dehydrating for 10min, and making xylene I and II solution transparent.
8) The clear paraffin sections were placed in a fume hood and sealed with neutral gum.
9) And then taken out for observation under a microscope and photographed to leave a photograph.
The results show that: trim55 compared to the respective sham group -/- Cardiac significantly increased in the-MI and C57-MI groups, significantly increased infarct size, significantly increased fibrosis in the infarct border zone, but Trim55 -/- The heart of MI is smaller than C57-MI, the extent of fibrosis in the infarcted border zone is significantly reduced (n=3, * p<0.05， ** p<0.01， *** p<0.001 (fig. 3D), prompt Trim55 -/- Can remarkably relieve fibrosis of the infarct border zone of mice.
(5) Western blot detects protein expression of fibrosis index (Collagen I and TGF-beta) in the border region of cardiac infarction.
See in particular example 1.Trim55 antibody (1:1000, U.S. Thermo), collagen I antibody (1:1000, U.S. CST), TGF-. Beta.antibody (1:1000, U.S. Abcam) and GAPDH antibody (1:1000, U.S. CST) were used as primary antibodies.
The results show that: trim55 compared to the respective sham group -/- Infarct border zone fibrosis was significantly increased in the-MI and C57-MI groups, but Trim55 -/- Infarct border zone fibrosis extent of MI was less than that of C57-MI group (n=3, * p<0.05， ** p<0.01， *** p<0.001 (fig. 3E-F), prompting Trim55 -/- Can remarkably relieve fibrosis of the infarct border zone of mice.
To sum up, whole body knockout Trim55 mice (Trim 55 -/- ) Reduce myocardial injury after MI.
Example 4: whole body knockout Trim55 mice (Trim 55) -/- ) Reduce myocardial cell apoptosis in the infarct border area after MI.
(1) Western blot detects protein expression of the heart infarct border zone apoptosis indicators (Bax, bcl-2 and clear caspase 3).
See in particular example 1.Trim55 antibody (1:1000, U.S. Thermo), bax antibody (1:1000, U.S. CST), bcl-2 antibody (1:1000, U.S. Abcam), clear caspase3 antibody (1:1000, U.S. CST) and GAPDH antibody (1:1000, U.S. CST) were used as primary antibodies.
The results show that: and each is associated withTrim55 compared to sham group -/- Infarct border zone apoptosis proteins Bax and clear caspase3 were significantly increased in the-MI group and C57-MI group, bcl-2 was significantly reduced, but Trim55 -/- Infarct border zone Bax and clear caspase3 of MI are significantly smaller than in the C57-MI group (n=3, * p<0.05， ** p<0.01， *** p<0.001 (fig. 4A-B), prompting Trim55 -/- Can obviously reduce the apoptosis of the infarct marginal zone of the mice.
(2) TUNEL staining detects the apoptosis in the border area of the heart infarction.
The TUNEL staining kit was used, the specific procedure was as follows.
1) Freezing tissue slices, rewarming for 10min at room temperature, placing the slices in a wet box, fixing with 85% -95% ethanol for 30min, and washing with PBS for 3 times, each for 3min.
2) 0.1% TritonX-100 was allowed to pass through for 15min and washed 3 times with PBS for 3min each.
3) TUNEL reaction was prepared and 50. Mu.L TdT+450. Mu.L fluorescein-labeled dUTP was mixed well, 50. Mu.L of each specimen was incubated at 37℃for 1h in the absence of light, and washed 3 times with PBS for 3min each.
4) DAPI dyes the cell nucleus, 5-10 min, PBS washes 3 times, each for 3min, and finally washes 3 times with distilled water.
5) Air drying, sealing with anti-quenching fluorescent sealing tablet, sealing with nail polish, and sealing with edge.
6) The photographs were observed under a microscope and photographed for storage.
The results show that: trim55 compared to the respective sham group -/- The number of infarct border zone apoptosis-positive cells was significantly increased in the-MI and C57-MI groups, but Trim55 -/- The number of infarct border zone apoptosis-positive cells of MI was significantly less than in the C57-MI group (n=3, * p<0.05 (fig. 4C), prompt Trim55 -/- Can obviously reduce the apoptosis of the infarct marginal zone of the mice.
To sum up, whole body knockout Trim55 mice (Trim 55 -/- ) Reduce myocardial cell apoptosis in the infarct border area after MI.
Example 5: systemic over-expression Trim55 mice (AAV-Trim 55) exacerbate post-MI myocardial injury.
(1) Western blot detects Trim55 expression in AAV-Trim55 mouse hearts.
See in particular example 1.Trim55 antibody (1:1000, thermo Inc. USA) and GAPDH antibody (1:1000, CST Co., USA) were used as primary antibodies.
The results show that: trim55 protein expression was significantly increased in AAV-Trim55 mouse hearts compared to AAV-CON mice (n=3, # p<0.05 (fig. 5A).
(2) And detecting Trim55 expression in AAV-Trim55 mouse hearts by fluorescent quantitative PCR.
See in particular example 1.
The results show that: trim55 mRNA expression was significantly increased in AAV-Trim55 mouse hearts compared to AAV-CON mice (n=3, # p<0.05 (fig. 5B).
(3) MI-28d, the mice survival curve was counted.
The results show that: the survival rates of both AAV-Trim55-MI group and AAV-CON-MI group were significantly reduced compared to the respective sham groups, but AAV-Trim55-MI survival rates were lower than AAV-CON-MI (n= 5,8,5, 10, p=0.1195) (fig. 5C).
(4) MI-28d, after mouse killing, the weight-to-heart ratio is counted.
See in particular example 3.
The results show that: the weight to weight ratio of both AAV-Trim55-MI group and AAV-CON-MI group was significantly increased, but the weight to weight ratio of AAV-Trim55-MI was significantly lower than AAV-CON-MI (n=5, * p<0.05， ** p<0.01， *** p<0.001 (fig. 5D), suggesting that the heart became larger after over-expression Trim55 aggravated MI.
(5) Mice were evaluated for cardiac contractile function by ultrasound.
See in particular example 3.
The results show that: the weight to weight ratio of both AAV-Trim55-MI and AAV-CON-MI groups was significantly reduced compared to the respective sham groups, but EF% and FS% of AAV-Trim55-MI were significantly lower than AAV-CON-MI, systolic LVAW was significantly lower than AAV-CON-MI (n=5, * p<0.05， ** p<0.01， *** p<0.001 (fig. 5E), suggesting that over-expression Trim55 may significantly exacerbate cardiac dysfunction in MI mice.
(6) HE staining detects myocardial infarction area and Masson staining detects the degree of myocardial fibrosis.
See in particular example 3.
The results show that: the hearts of the AAV-Trim55-MI group and AAV-CON-MI group were significantly increased, infarct size was significantly increased, infarct border zone fibrosis was significantly aggravated, but the hearts of AAV-Trim55-MI were larger than AAV-CON-MI, infarct border zone fibrosis degree was aggravated (n=3, * p<0.05， ** p<0.01， *** p<0.001 (fig. 5F), suggesting that over-expression of Trim55 may significantly increase the infarct extent in MI mice.
(7) Western blot detects protein expression of fibrosis index (Collagen I and TGF-beta) in the border region of cardiac infarction.
See in particular example 1.Trim55 antibody (1:1000, U.S. Thermo), collagen I antibody (1:1000, U.S. CST), TGF-. Beta.antibody (1:1000, U.S. Abcam) and GAPDH antibody (1:1000, U.S. CST) were used as primary antibodies.
The results show that: the infarct border region fibrosis was significantly increased in the AAV-Trim55-MI group and AAV-CON-MI group compared to the respective sham groups, but the infarct border region fibrosis of AAV-Trim55-MI was greater than in the AAV-CON-MI group (n=2-3, * p<0.05， ** p<0.01 (fig. 5G-H), suggesting that AAV-Trim55 may significantly exacerbate mouse infarct border zone fibrosis.
In conclusion, systemic over-expressed Trim55 mice (AAV-Trim 55) aggravate post-myocardial infarction myocardial injury.
Example 6: systemic over-expression Trim55 (AAV-Trim 55) mice exacerbate post-MI infarct border zone cardiomyocyte apoptosis.
(1) Western blot detects protein expression of the heart infarction border area apoptosis index (Bax).
See in particular example 1.Trim55 antibody (1:1000, U.S. Thermo Co.), bax antibody (1:1000, U.S. CST Co.) and GAPDH antibody (1:1000, U.S. CST Co.) were used as primary antibodies.
The results show that: AAV-Trim55-MI group and AAV-CON-MI group infarct side compared to the respective sham groupThe border region apoptotic protein Bax was significantly increased, and the infarct border region Bax of AAV-Trim55-MI was significantly more than that of AAV-CON-MI group (n=3, * p<0.05， ** p<0.01 (fig. 6A-B), suggesting that over-expression Trim55 may significantly exacerbate apoptosis in the infarct border zone in mice.
(2) TUNEL staining detects the apoptosis in the border area of the heart infarction.
See in particular example 4.
The results show that: the number of infarct border zone apoptosis-positive cells was significantly increased in AAV-Trim55-MI and AAV-CON-MI groups compared to the respective sham groups, but significantly more in AAV-Trim55-MI than in AAV-CON-MI group (n=3, * p<0.05， *** p<0.001 (fig. 6C), suggesting that over-expression Trim55 may significantly exacerbate apoptosis in the infarct border zone of mice.
In conclusion, whole-body over-expression Trim55 mice (AAV-Trim 55) aggravate myocardial cell apoptosis in infarct border areas after myocardial infarction.
Example 7: hypoxia-induced apoptosis of cardiomyocytes was promoted following cytologic administration of overexpressed Trim 55.
(1) Western blot detects expression of apoptosis proteins (clear caspase3, clear caspase8, clear caspase9 and Bcl-2) after overexpression of Trim55 in H9C2 cells.
See in particular example 1.Trim55 antibody (1:1000, U.S. Thermo), bcl-2 antibody (1:1000, abcam, U.S. Co.), clear caspase3 antibody (1:1000, CST, U.S. Co.), clear caspase8 antibody (1:1000, CST, U.S. Co.), clear caspase9 antibody (1:1000, CST, U.S. Co.), and GAPDH antibody (1:1000, CST, U.S. Co.) were used as primary antibodies.
The results show that: after Trim55 over-expression, the protein expression of clear caspase3 and of the exogenous apoptotic pathway clear caspase8 was significantly increased, whereas the protein expression of the endogenous apoptotic pathway clear caspase9 and Bcl-2 was not significantly altered (n=3, # p<0.05 (fig. 7A-B). It is suggested that Trim55 overexpression may promote apoptosis.
(2) Western blot detection of Trim55 over-expression in H9C2 cells followed by CoCl administration 2 After stimulationTrim55 and expression of apoptosis proteins (clear caspase3 and clear caspase 8).
See in particular example 1.Trim55 antibody (1:1000, U.S. Thermo), clear caspase3 antibody (1:1000, U.S. CST), clear caspase8 antibody (1:1000, U.S. CST) and GAPDH antibody (1:1000, U.S. CST) were used as primary antibodies.
The results show that: trim55 overexpression and CoCl administration 2 After that, the protein expression of clear caspase3 and clear caspase8 was further significantly increased (n=3, * p<0.05， ** p<0.01， *** p<0.001 (fig. 7C-D). Trim55 overexpression was suggested to promote hypoxia-induced apoptosis.
(3) Flow cytometry detection of H9C2 cells over-expressing Trim55 followed by CoCl administration 2 Apoptosis conditions.
The apoptosis flow kit is used, and the specific steps are as follows.
1) Cells were treated in groups and collected for flow detection after stimulation time.
2) Collecting cells: the supernatants of the test cells were collected separately in 15mL EP tubes, the cells were rinsed with 2mL PBS, 2mL pancreatin without EDTA was added, and the cells were digested in an incubator at 37℃until the cell mass was detached from the bottom of the dish. After termination of digestion with the supernatant just collected, transfer to the just 15mLEP tube. 2000rpm/s, room temperature, 5min, discard supernatant.
3) Washing the cells: cells were resuspended in 1mL of room temperature PBS and transferred to 1.5mL EP tubes. 2000rpm/s, room temperature, 5min, discard supernatant. And then washed once again with 1x binding buffer.
4) Cell count: cell counting was performed using a cytometer, 2X 10 for each group 6 And (3) cells.
5) Dyeing: cells were resuspended using 200. Mu.L of 1x binding buffer (2X 10) 6 ) All experimental groups required double staining with negative control and positive control required single staining tubes with two dyes. That is, 5. Mu.L of Annexin V dye was added to the double-dyeing tube, 5. Mu.L of 7-AAD, 5. Mu.L of Annexin V dye alone or 5. Mu.L of 7-AAD was added to the single-dyeing tube, and the name of the single-dyeing dye was clearly known.The mixture was flicked by hand and dyed in dark place for 15min. ( The flow tube and filter membrane were prepared during the staining process. Opening flow cytometer to check sheath fluid and waste liquid condition )
6) After the completion of the staining, 1mL of PBS at 2000rpm/s was added, and the mixture was centrifuged at room temperature for 5min, and the supernatant was discarded. Then 100. Mu.L PBS was added for resuspension, and the mixture was checked on the machine and plotted.
The results show that: coCl administration to control group 2 After that, the apoptosis level was significantly increased and the over-expressed Trim55 group was given CoCl 2 After that, the apoptosis level was further increased (n=3, * p<0.05， ** p<0.01 (fig. 7E). Trim55 overexpression was suggested to promote hypoxia-induced apoptosis.
(4) TUNEL staining detection of H9C2 cells over-expressing Trim55 followed by CoCl administration 2 Apoptosis conditions
The TUNEL kit was used as follows:
1) Using a cell slide, after discarding the culture supernatant, PBS was washed 3 times, fixed with 1mL paraformaldehyde for 30min, and PBS was washed 3 times.
2) 1mL of 0.1% TritonX-100 was allowed to pass through for 5-10 min and washed 3 times with PBS.
3) TUNEL reaction was prepared as described in example 4 (2), 50. Mu.L of each pellet was incubated at 37℃for 1h in the absence of light and washed 3 times with PBS.
4) DAPI stained nuclei were found in example 4 (2), washed 3 times with PBS and then 3 times with distilled water.
5) And (5) airing the sealing sheet, photographing under a microscope, and storing the photo.
The results show that: coCl administration to control group 2 After that, the number of apoptosis positive cells was significantly increased and the over-expressed Trim55 group was given CoCl 2 After that, the number of apoptosis-positive cells was further increased (n=3, ** p<0.01 (fig. 7F). Trim55 overexpression was suggested to promote hypoxia-induced apoptosis.
Taken together, H9C2 cells promote hypoxia-induced apoptosis following administration of overexpressed Trim 55.
While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (10)
- Use of a trim55 protein or an active fragment thereof for the preparation of a medicament for the prevention and/or treatment of heart failure due to abnormal heart remodeling caused by excessive apoptosis of cardiomyocytes after MI.
- 2. Use of Trim55 protein or an active fragment thereof according to claim 1, wherein the pharmaceutical composition comprises an inhibitor of Trim55 protein or an active fragment thereof.
- 3. Use of a nucleic acid molecule encoding a Trim55 protein or an active fragment thereof, a recombinant vector expressing a Trim55 protein or an active fragment thereof or a recombinant cell for the preparation of a medicament for the prevention and/or treatment of heart failure due to abnormal heart remodeling caused by excessive apoptosis of cardiomyocytes after MI.
- 4. The use according to claim 3, wherein the pharmaceutical composition comprises a nucleic acid molecule encoding a Trim55 protein or an active fragment thereof, a recombinant vector expressing a Trim55 protein or an active fragment thereof, or an inhibitor of a recombinant cell.
- 5. Use of an agent capable of inhibiting upregulation of Trim55 protein or active fragment thereof expression or promoting downregulation of Trim55 protein or active fragment thereof expression in the manufacture of a medicament for the prevention and/or treatment of heart failure due to abnormal heart remodeling resulting from excessive apoptosis of cardiomyocytes after MI.
- 6. A pharmaceutical composition for preventing and/or treating heart failure due to abnormal heart remodeling caused by excessive apoptosis of cardiac myocytes after MI, characterized in that the pharmaceutical composition comprises one or more of the following active ingredients:(1) An agent capable of inhibiting upregulation of Trim55 protein or active fragment thereof expression;(2) An agent that promotes down-regulation of Trim55 protein or an active fragment thereof;(3) A nucleic acid molecule encoding a Trim55 protein or an active fragment thereof, a recombinant vector or a recombinant cell expressing a Trim55 protein or an active fragment thereof.
- 7. The pharmaceutical composition of claim 6, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient; the dosage form of the pharmaceutical composition includes a pharmaceutically acceptable dosage form.
- 8. A pharmaceutical composition comprising a Trim55 protein or an active fragment thereof, a nucleic acid molecule encoding a Trim55 protein or an active fragment thereof, a recombinant vector or recombinant cell expressing a Trim55 protein or an active fragment thereof, or an agent capable of inhibiting down-regulation of Trim55 protein or an active fragment thereof, and optionally a pharmaceutically acceptable carrier or excipient; the composition is used for preventing and/or treating heart failure caused by abnormal heart remodeling caused by excessive apoptosis of cardiac muscle cells after MI.
- 9. Use of a reagent for detecting the expression level of Trim55 protein or an active fragment thereof in the preparation of a kit for the prediction of myocardial cell excessive apoptosis and/or the evaluation of the therapeutic effect and prognosis after MI.
- 10. A kit for detecting excessive apoptosis of myocardial cells after MI, comprising reagents for detecting expression levels of Trim55 protein or an active fragment thereof.
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