CN116509874A - Application of methylbardoxolone in preparation of medicine for improving viral myocarditis or viral myocardial injury - Google Patents
Application of methylbardoxolone in preparation of medicine for improving viral myocarditis or viral myocardial injury Download PDFInfo
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- CN116509874A CN116509874A CN202310049322.4A CN202310049322A CN116509874A CN 116509874 A CN116509874 A CN 116509874A CN 202310049322 A CN202310049322 A CN 202310049322A CN 116509874 A CN116509874 A CN 116509874A
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- viral
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- myocardial injury
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- myocarditis
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an application of methylbardoxolone in preparing a medicament for improving viral myocarditis or viral myocardial injury. The research of the invention discovers that the methyl bardoxolone can inhibit the activation of the inflammatory corpuscles of the NLRP3 in the cardiac muscle, reduce the content of myocardial injury markers CK-MB and cTnI in serum and inhibit the change of myocardial structure and function caused by virus infection, thereby realizing the improvement of viral myocarditis or viral myocardial injury and providing theoretical support for developing new medicaments for treating the viral myocarditis or the viral myocardial injury.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of methylbardoxolone in preparation of a medicament for improving viral myocarditis or viral myocardial injury.
Background
Viral myocarditis is a common myocardial inflammatory disease caused by virus infection, and seriously endangers the life safety of patients. Viral myocarditis causes a series of immune responses in infected individuals and is also an important cause of dilated cardiomyopathy.
Patients with viral myocarditis often develop various arrhythmias and impaired cardiac function, and even sudden cardiac death may occur, and endocardial biopsy is the gold standard for diagnosis of the disease. Endomembrane biopsies, however, are not popular in clinical work as an invasive test. The diagnosis of viral myocarditis still depends on medical history, clinical manifestation and related clinical examination indexes, especially cardiac magnetic resonance imaging examination, and is an important examination means for diagnosing viral cardiomyopathy. At present, a definite and effective treatment means for viral myocarditis is still lacking.
Oleanolic acid is a triterpene compound extracted from olive leaf and having antiinflammatory, liver protecting and antitumor effects, and can be used for treating liver injury, liver fibrosis, etc. 2 ⁃ cyano ⁃, 12 ⁃ dioxy oleanane ⁃ 1,9 (11) ⁃ dien ⁃ 28 ⁃ methyl ester (bardoxolone methyl, CDDO ⁃ Me), i.e. methyl bardoxolone, is a semisynthetic oleanolic acid derivative. CDDO-Me is used as the derivative of oleanolic acid, and has more remarkable anti-inflammatory, antioxidant and antitumor effects. At present, CDDO-Me is reported to be used for treating chronic kidney disease caused by type 2 diabetes, for example, patent application No. 201610329985.1 discloses an oleanolic acid derivative. CDDO-Me has also been reported to be useful in the treatment of pulmonary hypertension, as in the case of the inhalation of CDDO ⁃ NO on the induction of monocrotaline pulmonary hypertension in rats (Wu Xuecheng et al, month 9 of 2022, university of Nanjing medical science, vol.42, 9). There are reports that CDDO-Me has potential safety hazard in heart and is toxic to cardiac myocytes, so there is no report that CDDO-Me methyl bardoxolone is used for treating myocarditis.
Disclosure of Invention
The invention aims to provide the application of the methylbardoxolone in preparing the medicines for improving the viral myocarditis or the viral myocardial injury. The invention aims to study the protective effect and mechanism of the methylbardoxolone in viral myocarditis.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention there is provided the use of methylbardoxolone (CDDO-Me) in the manufacture of a medicament for ameliorating viral myocarditis or viral myocardial injury.
Preferably, the viral myocarditis or viral myocardial injury is viral myocarditis or viral myocardial injury caused by an infection with a cardiophilic virus.
Preferably, the cardiophilic virus is coxsackie B3 virus (CVB 3).
In a second aspect of the present invention, there is provided a medicament for treating viral myocarditis or viral myocardial injury, which comprises 0.5mg/ml of methylprednisolone as an active ingredient.
In a third aspect of the invention there is provided the use of methylbardoxolone in the manufacture of a medicament for reducing the content of CK-MB and cTnI in serum.
In a fourth aspect of the invention there is provided the use of methylbardoxolone in the manufacture of a medicament for inhibiting alterations in myocardial structure and function.
The invention has the beneficial effects that:
according to the research of the invention, the methylbardoxolone can activate Nrf2/HO-1 signal path, inhibit activation of NLRP3 inflammatory corpuscle, reduce the content of myocardial necrosis markers CK-MB and cTnI in serum, and inhibit myocardial structural and functional changes, thereby realizing improvement of viral myocarditis or viral myocardial injury, and providing theoretical support for developing new medicaments for treating viral myocarditis or viral myocardial injury.
Drawings
Fig. 1: the mice survival curve graph A is the survival curve of four groups of mice, B is the statistical analysis of the survival curves of the mice in the model group and the experimental group, and C is the weight line graph of the whole experimental process of the four groups of mice;
fig. 2: a content graph of myocardial injury markers in mouse serum, wherein A is a content statistical analysis graph of creatine kinase isozymes (CK-MB) in the mouse serum of each group measured by an Elisa method, and B is a content statistical analysis graph of troponin I (cTnI) in the mouse serum of each group measured by the Elisa method; values are expressed as mean ± standard error (x±sem), n=8, significance is expressed as p < 0.05; ns: p > 0.05,: p < 0.05,: p < 0.01:,: p < 0.001,: p < 0.0001;
fig. 3: CDDO ⁃ Me schematic for inhibiting myocardial remodeling, a is a mouse heart picture, B is a mouse myocardial HE staining slice (200X), C is a mouse heart ultrasound screenshot, D-H is a mouse heart Left Ventricular Ejection Fraction (LVEF), left ventricular foreshortening Fraction (FS), left anterior wall thickness (LVAWT), left ventricular end diastolic diameter (LVDd), and left ventricular end systolic diameter (LVDs); values are expressed as mean ± standard error (x±sem), n=9, significance is expressed as p < 0.05; ns: p > 0.05,: p < 0.05,: p < 0.01:,: p < 0.001,: p < 0.0001;
fig. 4: a is an immunofluorescence Image (IFC) of NLRP3 expressed in mouse cardiac muscle, B is a fluorescence image of Caspase-1 expressed in mouse cardiac muscle, C is an immunofluorescence image of ASC expressed in mouse cardiac muscle, and D is an NLRP3, caspase-1 and ASC western blot image in mouse cardiac muscle;
fig. 5: a is a statistical analysis chart of the content of the intramyocardial NLRP3 measured by a WB experiment; b is a statistical analysis chart of the content of the intramyocardial NLRP3 related mRNA measured by a PCR experiment, C is a statistical analysis chart of the content of the intramyocardial NLRP3 measured by an IFC experiment;
fig. 6: a is a statistical analysis chart of content of Caspase-1 in cardiac muscle measured by WB experiment; b is a statistical analysis chart of the content of the related mRNA of the Caspase-1 in the cardiac muscle measured by a PCR experiment, C is a statistical analysis chart of the content of the Caspase-1 in the cardiac muscle measured by an IFC experiment;
fig. 7: a is a statistical analysis chart of the content of ASC in cardiac muscle measured by WB experiment; b is a statistical analysis chart of the content of the ASC related mRNA in the cardiac muscle measured by a PCR experiment, C is a statistical analysis chart of the content of the ASC in the cardiac muscle measured by an IFC experiment;
fig. 8: a is a statistical analysis chart for measuring the IL-1 beta content in the serum of the mouse by a Luminex method, and B is a statistical analysis chart for measuring the IL-1 beta related mRNA content in the myocardium of the mouse by a PCR method;
fig. 9: a is a statistical analysis chart of IL-18 content in serum of a mouse measured by an Elisa method, and B is a statistical analysis chart of IL-18 related mRNA content in myocardial of the mouse measured by a PCR method;
fig. 10: a is a statistical analysis chart of IL-6 content in mouse serum measured by a Luminex method, B is a statistical analysis chart of IL-10 content in mouse serum measured by a Luminex method, C is a statistical analysis chart of TNF-alpha content in mouse serum measured by a Luminex method,
in fig. 5 to 10, WB and PCR experiments each group n=3, luminex and Elisa experiments each group n=8, ifc experiments each group n=6, statistics expressed as mean ± standard error (x±sem), significance expressed as p < 0.05; ns: p > 0.05,: p < 0.05,: p < 0.01:,: p < 0.001,: p < 0.0001;
fig. 11: a is immunofluorescence map (IFC) of Nrf2 expression in mouse myocardium, B is immunofluorescence map of p-Nrf2 expression in mouse myocardium, C is immunofluorescence map of HO-1 expression in mouse myocardium, D is immunofluorescence map of NQO1 expression in mouse myocardium, E is western blot map (WB) of Nrf2, p-Nrf2, HO-1 and NQO1 in mouse myocardium, wherein WB and PCR experiments are each set n=3, luminex and Elisa experiments are each set n=8, IFC experiments are each set n=6, statistics are expressed as mean ± standard error (x±sem), and significance is expressed as p < 0.05; ns: p > 0.05,: p < 0.05,: p < 0.01:,: p < 0.001,: p < 0.0001;
fig. 12: a is a statistical analysis chart of the content of Nrf2 in the myocardium measured by a WB (reverse transcription) experiment, B is a statistical analysis chart of the content of mRNA (messenger ribonucleic acid) related to the Nrf2 in the myocardium measured by a PCR (polymerase chain reaction) experiment, and C is a statistical analysis chart of the content of the Nrf2 in the myocardium measured by an IFC (inverse transformation) experiment;
fig. 13: a is a statistical analysis chart of the content of the p-Nrf2 in the myocardium measured by a WB (reverse transcription) experiment, and B is a statistical analysis chart of the content of the p-Nrf2 in the myocardium measured by an IFC (inverse transformation) experiment;
fig. 14: a is a statistical analysis chart of HO-1 content in mouse cardiac muscle measured by a WB method, B is a statistical analysis chart of HO-1 related mRNA content in mouse cardiac muscle measured by a PCR method, and C is a statistical analysis chart of HO-1 content in mouse cardiac muscle measured by an IFC method;
fig. 15: a is a statistical analysis chart of the NQO1 content in the myocardium of the mouse measured by a WB method, B is a statistical analysis chart of the NQO1 related mRNA content in the myocardium of the mouse measured by a PCR method, and C is a statistical analysis chart of the NQO1 content in the myocardium of the mouse measured by an IFC method;
in fig. 12 to 15, WB and PCR experiments each set n=3, luminex and Elisa experiments each set n=8, ifc experiments each set n=6, statistics expressed as mean±standard error (x±sem), significance expressed as p < 0.05; ns: p > 0.05,: p < 0.05,: p < 0.01:,: p < 0.001,: p < 0.0001.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, 2 ⁃ cyano ⁃, 12 ⁃ dioxyoleanane ⁃ 1,9 (11) ⁃ dien ⁃ 28 ⁃ methyl ester (bardoxolone methyl, CDDO ⁃ Me), i.e., methylbardoxolone, has the structural formula:
currently, CDDO ⁃ Me is mainly used for the treatment of chronic kidney disease, pulmonary hypertension, and tumors and cancers. The U.S. FDA has granted CDDO-Me an orphan drug for the treatment of Alport syndrome and pulmonary arterial hypertension, but there is a research statement that CDDO ⁃ Me presents a safety hazard in the heart and is toxic to cardiomyocytes. So there is no report on the use of CDDO-Me methyl bardoxolone in treating myocarditis.
Based on this, the object of the present invention is to provide the use of methylbardoxolone in the preparation of a medicament for ameliorating viral myocarditis or viral myocardial injury. Viral myocarditis can lead to changes in myocardial structure, and can lead to decline in cardiac function and various arrhythmias, and even sudden cardiac arrest. However, the disease is basically an inflammatory disease associated with viruses, followed by an immune response and subsequent lesions.
NLRP3 inflammatory corpuscles are a complex of proteins comprising three parts NLRP3, ASC and pro-caspase-1, which complex activates caspase-1 and thus mediates maturation and secretion of pro-inflammatory cytokines such as IL-1 beta and IL-18. Activation of NLRP3 inflammatory bodies is closely related to microbial infection of bacteria, viruses, and the like. After virus infection, the inflammatory corpuscles are activated, and the inflammatory factors IL-1 beta and IL-18 are further activated after the activation of the inflammatory corpuscles, so that subsequent inflammatory reactions are caused. Nrf2 is an important anti-inflammatory and antioxidant factor in the body and has important protection effect on the body. Nrf2 plays a very important anti-inflammatory role in a variety of diseases. The research of the invention discovers that CDDO-Me not only has the traditional anti-inflammatory mode of reducing the expression of inflammatory factors TNF-alpha, IL-6 and IL-10 by activating Nrf2/HO-1 signal paths, but also can inhibit the activation of inflammatory bodies of NLRP3 in cardiac muscle, reduce the expression of inflammatory factors IL-1 beta and IL-18 and further play an anti-inflammatory role. After viral myocarditis is affected, particularly in the immune activation period, myocardial structure can be changed, and the symptoms such as edema, necrosis, massive inflammatory cell infiltration and the like are presented, so that CDDO-Me can inhibit myocardial structure change and inflammatory cell infiltration; thus, the method for treating myocarditis or myocardial injury caused by Coxsackie B3 virus provides theoretical support for developing new medicaments for treating viral myocarditis or viral myocardial injury.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
Description: CVB3 virus was purchased from Shandong national academy of medical science; the applicant holds this strain and is willing to issue to the public within 20 years from the filing date in accordance with the patent statute.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available.
Examples
1. Test method
1.1 chemical reagents and Main instruments
0.25% trypsin solution (Invitrogen, U.S.), BCA protein quantification kit (Biyun Biotechnology Co., ltd., china), CDDO-Me (Med Chem Express, U.S. Pat.), ECL luminescence kit (Shanghai Seisakusho Biotech Co., ltd.), fastPure cell/tissue total RNA extraction kit (Nanno Biotechnology Co., ltd.), HE dye liquor kit (Biyun Biotechnology Co., ltd., china), hiScript III All-in-one RT SuperMix Perfect for qPCR (Nanno Biotechnology Co., ltd.), RIPA lysate (strong) (Biyun Biotechnology Co., ltd., china), mouse CK-MB, cTn-I, IL-1β, IL-6, IL-10, IL-18, TNF- α ELISA kit (Wuhan Iridere Biotechnology Co., ltd.), anti-ASC antibody (U.S. CellSignaling Technology), anti-Caspase 1 antibody (Wuhan Sanying, china), anti-HO-1 antibody (UK abcam Co., ltd.), anti-NLRP 3 antibody (U.S. Thermosher), anti-NQO 1 antibody (UK abcam Co., ltd.), anti-Nrf 2 antibody (U.S. Thermosher Co., ltd.), anti-p-Nrf 2 antibody (U.S. Thermosher), anti- β -actin antibody (Sungene Biotechnology, china), primer design synthesis (Jinan Bos Biotechnology Co., ltd.), fluorescent secondary antibody (Wuhan Sieve Biotechnology Co., ltd.), spontaneous fluorescence quencher (Hansal Sieve Biotechnology Co., ltd), vevo2100 small animal ultrasound imaging System (Fuji Visual Sonics Co.).
1.2 laboratory animals and methods
After taking 72 male SPF-grade Balb/c mice (purchased from Experimental animal technologies Co., ltd. In Violet, beijing) of 6 weeks old and adaptively feeding for 1 week, they were randomly divided into 4 groups: blank, drug only (CDDO-Me injection), model (CVB 3 virus injection) and experimental (CVB 3 virus+CDDO-Me injection) groups of 18 animals each. CVB3 Virus solution (10) 8 TCID 50) is diluted by PBS buffer solution for 10 times to obtain virus injection, and a model group and an experimental group establish a viral myocarditis model according to 100 mu L/single intraperitoneal injection of the virus injection on day 0; the blank and drug alone groups were given 100 μl/PBS buffer for intraperitoneal injection as controls.
From day 1, the CDDO-Me intraperitoneal injection was administered at 5mg/kg/24h in the drug alone group and the experimental group at a concentration of 0.5mg/mL and a volume of 10mL/kg; the control group and the model group were given the same volume of physiological saline for intraperitoneal injection once a day for 10 consecutive days. Mice were examined daily for body weight and status. Body weight and survival curves were recorded on day 10 and surviving mice were perfected with heart color ultrasound. And then, the mice are subjected to anesthesia treatment by intraperitoneal injection of pentobarbital sodium (50 mg/kg), when the anesthesia state is satisfactory, the chest cavity is opened to expose the heart, blood is taken from the apex region, and serum is taken and stored at the temperature of minus 80 ℃. Taking hearts, washing residual blood and broken tissues by using normal saline, dividing each heart into two parts, soaking one part in 4% paraformaldehyde, quick-freezing the other part in liquid nitrogen, and then freezing at-80 ℃ for later use as a subsequent experiment.
1.3 cardiac ultrasound examination
The mice were removed from their chest bristles, the skin exposed, and 1.5% isoflurane was administered for continued inhalation anesthesia, and the anesthetic gas flow rate was adjusted according to the anesthetic state. The heart color ultrasound examination was completed on mice using a Vevo2100 small animal ultrasound imaging system (Fuji Visual Sonics Co.). Left ventricular end systolic inner diameters (LVDs), left ventricular end diastolic inner diameters (LVDd), left anterior wall thickness (LVAWT), left Ventricular Ejection Fraction (LVEF), and left ventricular short axis shortening (FS) were measured and recorded. Each group of mice was evaluated for cardiac structure and function.
1.4 serological examination
Thawing the serum for later use. Detecting serum IL-18 levels in mice using a Elabscience Elisa kit; the levels of IL-1 beta, TNF-alpha, IL-6 and IL-10 in the serum of mice are detected by using a Luminex liquid phase suspension chip, and the operations are strictly carried out according to the specification.
1.5 Western blotting experiments
The frozen heart tissue is taken out, redundant tissue is excised, the cardiac muscle is cut into the size of 1mm, and the heart muscle is washed twice by precooled PBS solution, so that residual blood and broken tissue are removed. The tissue was ground as finely as possible using a mortar and liquid nitrogen. The ground heart tissue was transferred into a 1.5ml ep tube, an appropriate amount of tissue lysate was added according to the instructions, and the mixture was put on ice for 30 minutes for lysis, and the supernatant was taken after centrifugation. The protein solution concentration of each sample was measured using BCA kit to prepare 2. Mu.g/. Mu.l protein solution. To SDS-PAGE gel, 20. Mu.g of the protein extract was added for electrophoresis, and the subsequent procedures were performed according to Western Blotting standard methods to determine protein levels of Nrf2, p-Nrf2, HO-1, quinone oxidoreductase 1 (NQO 1) and NLRP3, apoptosis-related spotted protein (ASC), and Caspase 1 (Caspase-1) in the proteins. And using ImageJ software to determine gray values, and using β -actin as an internal reference protein for standardized comparison.
1.6RT-PCR experiments
The myocardial tissue was treated and ground as described above, and the total RNA extract was added to prepare an RNA extract, and the concentration was measured. Each sample was taken at 500ng, reverse transcribed into C-DNA by RT-PCR kit, amplified, and the relative expression levels of mRNA of myocardial tissues Nrf2, HO-1, NQO1 and NLRP3, ASC, caspase-1, IL-lβ, IL-18 were quantified by real-time fluorescence. Primer design was done by atanan bosch biotechnology limited.
1.7 hematoxylin-eosin staining and immunofluorescence experiments
Heart tissue immersed in 4% paraformaldehyde solution in advance was paraffin-embedded to prepare paraffin sections of 5 μm thickness. Hematoxylin-eosin (HE) staining was performed and myocardial tissue structure changes were observed under the light microscope. Immunofluorescence experiments are carried out, images are observed and acquired under a fluorescence microscope, and protein levels of Nrf2, p-Nrf2, HO-1, NQO1, NLRP3, ASC and caspase-1 in myocardial tissues of the mice are quantitatively analyzed through imageJ software.
1.8 statistics and analysis
Data were counted and analyzed using Graphpad Prism9 software and measured data were expressed as mean ± standard deviation (x±s). The normal distribution and variance homogeneity of each group of data are checked, and the difference and significance of each group of data are compared by applying single factor variance analysis, wherein the significance is expressed as P < 0.05.
2. Conclusion(s)
2.1CDDO-Me increased survival and improved weight status in mice with viral myocarditis
As shown in FIG. 1, the survival rate of CVB3 mice is only 50% in ten days, which is far lower than 100% of that of the control group and CDOO-Me group, while the survival rate of experimental mice is 72.2% which is higher than that of the model group, and the survival rate is statistically significant. As shown in fig. 1C, the average body weight of the mice in the model group was lower than that in the control group by the day 2 of the experiment, and the average body weight of the mice in the experimental group was slightly higher than that in the model group but still lower than that in the control group.
2.2 CDDO-Me reduces myocardial injury marker levels
CK-MB and cTnI are used as markers of myocardial injury and are clinically used for assisting in evaluating the degree of myocardial injury and recovery condition of viral myocarditis. As shown in fig. 2, serum central muscle injury markers were significantly elevated after mice injected with CVB3, and myocardial injury was reduced after CDOO-Me intervention. As can be seen from the distributions in FIGS. 2A and 2B, the serum of mice in CVB3 group, i.e. model group, is significantly increased in CK-MB and cTnI compared with the control group, and has statistical significance. After CDDO-Me treatment, both myocardial necrosis markers were significantly reduced compared to the model group.
2.3CDD0-Me reduces myocardial remodeling
After viral myocarditis is suffered from diseases, particularly in the immune activation period, myocardial structures can be changed, myocardial edema, dissolved necrosis, massive inflammatory cell infiltration and the like appear, and thus ventricular enlargement and heart contraction function decline appear. As shown in fig. 3, CDDO-Me reduced myocardial remodeling in mice due to CVB3 infection. As can be seen in fig. 3A, the heart of the mice in the control group and the mice in the group injected with CDDO-Me alone is normal in shape and smooth in surface; the hearts of the disease model group and the experimental group are increased, the epicardial surface is not smooth, and a large amount of white spots can be seen. FIG. 3B shows the stained sections of mouse myocardial HE, the Control group and CDDO-Me group mouse myocardial cells are normal in size, uniform in morphology, regular in myocardial fiber arrangement, uniform and dense in distribution, clear in myofiber structure, and no obvious inflammatory cell infiltration is seen in the stroma. In contrast, mice in group CVB3 and group CVB3+CDDO-Me had cardiomyocytes of different sizes and non-uniform morphology, and were seen with varying degrees of cardiomyocyte edema, lysis necrosis, myofiber alignment disorder, and a part of cardiomyocytes were seen with vacuolated changes accompanied by infiltration of a large number of interstitial inflammatory cells. Compared with CVB3 group, CVB3+CDDO-Me group has reduced myocardial cell edema and necrosis proportion, and reduced inflammatory cell infiltration, which indicates that myocardial inflammation is inhibited. Fig. 3D-H show statistical analysis results of cardiac ultrasound, with significantly reduced left ventricular ejection fraction and left ventricular shortening fraction in the CVB mice compared to the control group, with significantly increased left ventricular diameter at end systole, while the experimental group significantly alleviated these changes, with statistical significance.
2.4 CDDO-Me reduces mouse intramyocardial NLRP3 inflammatory corpuscles and inflammatory factors downstream thereof
Viral myocarditis is an inflammatory disease associated with viral infection and manifests itself as inflammatory cell infiltration of the myocardium. This example demonstrates that CDDO-Me treatment inhibits activation of NLRP3 inflammatory bodies and expression of inflammatory factors downstream thereof in myocarditis mice caused by CVB3 infection. As shown in FIG. 4, the serum or intramyocardial NLRP3 inflammatory corpuscle and related inflammatory factor level of the mice after CVB3 infection is increased, and the inflammatory factor level is reduced to different degrees after CDDO-Me treatment, so that the method has statistical significance. The WB experiments prove that the myocardial NLRP3, caspase-1 and ASC content of the mice in the model group are higher than those of the mice in the control group (figures 4D, 5A, 6A and 7A), the expression of mRNA related to three proteins is also obviously higher than that of the mice in the control group (figures 5B, 6B and 7B), and the immunofluorescence experiments prove that the phenomenon is also proved (figures 4A, 4B and 4C). Serum levels of the remaining inflammatory factors activated by NLRP3 inflammatory bodies were also higher in the model group than in the control group. IL-1. Beta. In the serum of each group of mice was measured by the Luminex method, and the IL-18 level in the serum of the mice was measured by the Elisa method, and the results of the model group were higher than those of the control group. The IL-1 beta and IL-18 related mRNA in the myocardium of the mice in the model group are obviously higher than those of the mice in the control group by the RT-PCR method. These inflammatory factors were reduced to different extents and were statistically significant after CDDO-Me treatment.
2.5 CDDO-Me increased protein levels of Nrf2 and HO-1 in mouse myocardium
As mentioned previously, nrf2 is an important anti-inflammatory substance in the body. Nrf2 enters the nucleus after activation, and can stimulate the synthesis of downstream anti-inflammatory substances such as HO-1 and NQO-1, thereby playing an anti-inflammatory role. And phosphorylation is an important link in Nrf2 activation for subsequent anti-inflammatory modulation.
Experiments prove that CDDO-Me can activate Nrf2/HO-1 signal paths and improve the levels of Nrf2, p-Nrf2, HO-1 and NQO1 in mice. The levels of the above proteins in mouse myocardium were determined by WB and IFC experiments (see FIGS. 11A, 11B, 11C, 11D, 11E and 12A, 12C, 13A, 13B, 14A, 14C, 15AB, 15C), while the levels of Nrf2, HO-1 and NQO 1-related mRNA in mouse myocardium were determined by RT-PCR (FIGS. 12B, 14B and 15B). From the results, the Nrf2/HO-1 signal pathway in the myocardium of the mice after CVB3 infection is inhibited, the content of the anti-inflammatory factors and the expression of related mRNA are reduced compared with the control group, and the Nrf2/HO-1 signal pathway is activated through CDDO-Me intervention, so that the anti-inflammatory effect is achieved, and the anti-inflammatory agent has statistical significance.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (6)
1. Use of methylbardoxolone in the manufacture of a medicament for ameliorating viral myocarditis or viral myocardial injury.
2. The use according to claim 1, wherein the viral myocarditis or viral myocardial injury is viral myocarditis or viral myocardial injury caused by an cardiotropic viral infection.
3. The use according to claim 1, characterized in that the cardiophilic virus is a coxsackie B3 virus.
4. A medicament for treating viral myocarditis or viral myocardial injury, which is characterized in that the medicament takes 0.5mg/ml of methylbardoxolone as an active ingredient.
5. Use of methylprednisolone in the preparation of a medicament for reducing CK-MB and cTnI content in serum.
6. Use of methylbardoxolone in the manufacture of a medicament for inhibiting alterations in myocardial structure and function.
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