CN114028371A - Application of alpha-ketoglutaric acid in preparation of medicine for treating myocardial infarction - Google Patents
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Abstract
The invention discloses application of alpha-ketoglutaric acid in preparing a medicament for treating myocardial infarction, wherein a metabolic intermediate product alpha-ketoglutaric acid used in the invention can improve the heart function reduction caused by myocardial infarction and reduce the myocardial infarction area by promoting the proliferation of adult myocardial cells. The specific mechanism is that alpha-ketoglutarate removes the inhibition of inhibiting transcriptional histone modification H3K27me3 on cell cycle regulatory genes by activating the activity of demethylase JMJD3 and enhancing the demethylation of H3K27me3, and finally promotes the proliferation of adult myocardial cells to fulfill the aims of repairing myocardial infarction and preventing and treating heart failure. The alpha-ketoglutaric acid provided by the invention can be used for treating myocardial infarction and has important clinical significance for treating myocardial infarction.
Description
Technical Field
The invention belongs to the field of biomedical research, and particularly relates to application of alpha-ketoglutaric acid in preparation of a medicament for treating myocardial infarction.
Background
Myocardial infarction is one of main diseases which are harmful to human health, and has the characteristics of high morbidity and poor prognosis. After myocardial infarction, a large number of myocardial cells are necrosed and apoptotic, and adult cardiac muscle cannot be repaired by self proliferation, so that the cardiac function is continuously deteriorated, and finally, heart failure is developed. The current methods of drug therapy, interventional therapy, coronary bypass, and the like adopted clinically are helpful for improving the symptoms of patients and recovering blood supply, but cannot fundamentally update and repair necrotic myocardium. Therefore, repair of cardiac function ultimately must also rely on myocardial regeneration. However, for a long time, the mammalian heart has been considered as a terminally differentiated organ, without regenerative capacity. However, recent tracking of C14 and the thymidine analog iododeoxyuridine in muscle cells has demonstrated the regenerative properties of human myocardium.
A large number of domestic and foreign researches use a lineage tracing method to trace the source of regenerated myocardium, and the result proves that the original cardiomyocyte proliferation is the source of the regenerated myocardium. However, adult mammals have an extremely limited ability to proliferate myocardium and are not sufficient to achieve myocardial regeneration. Therefore, there is a need for a drug that can effectively promote proliferation of adult cardiomyocytes and further promote regeneration of myocardium to achieve treatment of myocardial infarction.
Alpha-ketoglutarate is an important intermediary metabolite in the tricarboxylic acid cycle, a weak acid containing two carboxyl groups and one keto group. Alpha-ketoglutarate is involved in the regulation of various cellular biological processes such as autophagy, inflammation, aging, and cell proliferation. Researches show that the alpha-ketoglutaric acid can remarkably promote the proliferation of vascular endothelial cells and skeletal muscle satellite cells. In addition, α -ketoglutarate also inhibits pathological myocardial hypertrophy by inhibiting autophagy. However, it has not been reported whether α -ketoglutarate can affect cardiomyocyte proliferation and can play a role in myocardial infarction.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an application of α -ketoglutaric acid in preparing a medicament for treating myocardial infarction, and an object of the present invention is to provide an application of a pharmaceutical composition in treating myocardial infarction.
In order to achieve the purpose, the invention provides the following technical scheme:
1. application of alpha-ketoglutaric acid in preparing medicine for treating myocardial infarction is provided.
As one of the preferred technical solutions, the treatment is specifically:
a. repairing cardiac function damage caused by myocardial infarction;
b. reducing scar area caused by myocardial infarction;
c. promoting proliferation of myocardial cells.
As one of the preferable technical solutions, the cardiac function includes left ventricular ejection fraction, left ventricular short axis shortening rate, left ventricular end-systolic volume and left ventricular end-diastolic volume.
As one of the preferred technical schemes, the mechanism of the alpha-ketoglutarate in treating myocardial infarction is as follows: through activating the activity of JMJD3, the inhibition of H3K27me3 in the cardiac muscle cells on the cell cycle is reduced, and the proliferation of the cardiac muscle cells is promoted.
2. The use of a pharmaceutical composition comprising said alpha-ketoglutarate and one or more pharmaceutically acceptable carriers or excipients in the treatment of myocardial infarction.
The invention has the beneficial effects that:
the invention discloses application of alpha-ketoglutaric acid in preparation of a medicament for treating myocardial infarction, and researches influence of the alpha-ketoglutaric acid on cardiac function damage caused by the myocardial infarction by constructing a C57 mouse myocardial infarction model. The experimental results confirm that the cell membrane permeability alpha-ketoglutaric acid (2-ketoglutaric acid dimethyl ester; DM-alpha-KG) can significantly reduce infarct size and improve cardiac function (left ventricular ejection fraction, left ventricular short axis shortening rate, left ventricular end-systolic volume and left ventricular end-diastolic volume). In addition, alpha-ketoglutarate significantly promotes proliferation of adult cardiomyocytes in developmental and infarct models. Thus alpha-ketoglutarate can reduce cardiac damage caused by myocardial infarction. The invention also verifies the influence of alpha-ketoglutaric acid on the proliferation of the myocardial cells in primary myocardial cells cultured in vitro, and is consistent with animal experiment results, and immunofluorescence experiment results show that the alpha-ketoglutaric acid can obviously increase the number of proliferation markers Ki67 and PH3 positive myocardial cells. These results indicate that alpha-ketoglutarate is indeed able to promote cardiomyocyte proliferation and repair of infarcted myocardium.
The invention also researches the molecular mechanism of promoting the proliferation of the myocardial cells by the alpha-ketoglutaric acid. The results show that alpha-ketoglutarate treatment can activate the activity of JMJD3 in cardiomyocytes. The results of the blocking experiments further show that the cardiomyogenesis promoting effect of α -ketoglutarate is blocked when JMJD3 in cardiomyocytes is knocked down. ChIP-sequencing analysis shows that alpha-ketoglutarate can activate the activity of demethylase JMJD3, so that the enrichment of transcription inhibition modified H3K27me3 on cell cycle genes is reduced, and the aims of promoting the proliferation of myocardial cells and repairing the infarcted myocardium are finally fulfilled.
The invention discovers for the first time that the alpha-ketoglutaric acid can be used for treating myocardial infarction (cardiac function damage and increase of cardiac scar area) and promoting proliferation of myocardial cells. The molecular mechanism is as follows: the alpha-ketoglutaric acid can release the inhibition of H3K27me3 on cell cycle regulatory genes by activating the activity of JMJD3 and enhancing the demethylation of H3K27me3, and finally promotes the proliferation of adult myocardial cells, thereby achieving the purposes of repairing myocardial infarction and preventing and treating heart failure. The invention firstly provides and proves that the alpha-ketoglutaric acid has the effect of treating myocardial infarction, not only provides a theoretical basis for adult myocardial proliferation and regeneration, but also provides a new idea for clinical treatment of myocardial infarction. In addition, alpha-ketoglutaric acid is a metabolite contained in a human body, and side effects caused by the alpha-ketoglutaric acid are relatively small, so that the alpha-ketoglutaric acid can be applied to medicines and health-care products.
Drawings
FIG. 1 shows the effect of exogenous α -ketoglutarate at different concentrations on the rate of cardiomyocyte proliferation (representative and statistical representation of cell proliferation marker Ki67 positive cardiomyocytes).
Fig. 2 shows the change in α -ketoglutarate levels after knockdown of α -ketoglutarate dehydrogenase (OGDH) in cardiomyocytes (P <0.05, n ═ 3 compared to control).
A, B in fig. 3 is the effect of increasing endogenous α -ketoglutarate levels in cardiomyocytes on cardiomyocyte proliferation (P <0.05, n ═ 3 compared to control).
Figure 4 shows the effect of α -ketoglutarate injection on body weight ratio of mouse hearts during development (× indicates P <0.05, n ═ 4 compared to control).
Figure 5 shows WGA immunofluorescence staining for cardiomyocyte size (P <0.05, n-4 compared to control).
Figure 6 shows EdU pulse tracking to examine the effect of α -ketoglutarate injection on cardiomyocyte proliferation during development (× indicates P <0.05, n ═ 4 compared to control).
Figure 7 is a graph of the effect of α -ketoglutaric acid on post-infarct cardiac function (× indicates P <0.05, n ═ 3 compared to control).
Figure 8 is the effect of α -ketoglutaric acid on myocardial infarct size (×) P <0.05, n ═ 3 compared to control.
Figure 9 shows EdU pulse tracking to determine the effect of α -ketoglutarate injection on adult cardiomyocyte proliferation (× indicates P <0.05, n ═ 3 compared to control).
FIG. 10 shows the expression level of H3K27me3 in heart measured by Western Blot (A: the expression level of H3K27me3 in heart of mice aged 1 day, 28 days and 28 days injected with alpha-ketoglutarate; B: the effect of knocking down PHGDH in cardiomyocytes and supplementing alpha-ketoglutarate on the expression level of H3K27me3 in cardiomyocytes; P <0.05, n ═ 3 as compared with control group).
Figure 11 shows PH3 immunofluorescence staining to detect cardiomyocyte proliferation (effect of supplementation with α -ketoglutarate on cardiomyocyte proliferation after knockdown of JMJD3 in cardiomyocytes;. indicates P <0.05 and n ═ 3 compared to control). )
Fig. 12 shows the level of enrichment of H3K27me3 at the start site of transcription of the genome (n-2).
Fig. 13 shows the level of enrichment of H3K27me3 on cell cycle-related genes CCND1, CDK1, CDK4 and CDK6 (n-2).
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1
Alpha-ketoglutaric acid can promote proliferation of primary cardiomyocytes cultured in vitro
The method for extracting the myocardial cells of the newborn SD rat comprises the following steps:
(1) 10ml of each of collagenase type II and trypsin solutions were prepared at a concentration of 0.08% and 0.125%, respectively.
(2) Ventricular tissue from the suckling mice was harvested by ophthalmic clipping and stored in a precooled Hanks buffer.
(3) Cutting the tissue to 1mm3The small pieces were then rinsed again with pre-cooled Hanks buffer solution.
(4) Sucking away the washing liquid, adding a proper amount of trypsin, gently and repeatedly blowing for 1 minute, and discarding the liquid after the tissues are gathered into a cluster.
(5) Collagenase was added, blown up and digested at 37 ℃ for 30 minutes.
(6) Repeatedly and gently blowing, after the tissue blocks are dissolved, adding lml fetal calf serum, and stopping digestion.
(7) The cell suspension is filtered by a 100-mesh (50um) cell sieve, the filtrate is placed in a centrifuge tube and centrifuged for 5 minutes at 4 ℃ and 1000rmp, the supernatant is discarded, and an appropriate amount of culture medium is added to resuspend the cells.
(8) And (3) differentially adhering to the wall for 2 hours, and discarding most of non-myocardial cells to obtain the myocardial cells of the mice and the suckling mice.
(9) Effect of exogenous α -ketoglutarate on cardiomyocyte proliferation.
After treating the cardiomyocytes with different concentrations of alpha-ketoglutaric acid (0.1, 1, 5mM), the proliferation of the cardiomyocytes was detected by Ki67 proliferation marker immunofluorescence staining. The results show (figure 1) that alpha-ketoglutarate can promote the proliferation of myocardial cells, and the effect is most obvious at 5 mM.
(10) Effect of endogenous alpha-ketoglutarate on cardiac cell proliferation.
siRNA knockdown alpha-ketoglutarate dehydrogenase (OGDH) in myocardial cells to increase endogenous alpha-ketoglutarate, and changes in the proliferation capacity of the myocardial cells of newborn mice are observed. The change of the content of alpha-ketoglutarate in the cells is detected by utilizing the alpha-ketoglutarate detection kit, and the result shows that (figure 2) knocking down the alpha-ketoglutarate dehydrogenase can increase the content of the alpha-ketoglutarate in the myocardial cells; in addition, the cell proliferation markers (Ki67, PH3) were detected by immunofluorescence staining, and the results showed (A, B in fig. 3) that the knock-down of α -ketoglutarate dehydrogenase promoted cardiomyocyte proliferation.
Example 2
Alpha-ketoglutaric acid can promote proliferation of mouse cardiac muscle cells in development stage
The 7-day-old mice were divided into experimental and control groups, and were subjected to intraperitoneal injection of α -ketoglutaric acid and PBS each day, and after 2 weeks, the heart-to-body ratio was evaluated, and the size of cardiomyocytes was further detected by Wheat Germ Agglutinin (WGA) staining. The results show that alpha-ketoglutarate injection increases the body weight ratio of the mouse heart (fig. 4), but no significant change in cardiomyocyte volume occurs (fig. 5). The expression of proliferation markers (EdU pulse markers) was detected by immunofluorescence staining, and as shown in FIG. 6, the proliferation of mouse cardiomyocytes was promoted by α -ketoglutarate injection.
Example 3
Alpha-ketoglutaric acid can improve post-infarct cardiac function and infarct size and promote proliferation of adult cardiomyocytes
(1) Constructing a mouse myocardial infarction model, dividing a mouse into an experimental group and a control group, respectively carrying out intraperitoneal injection on alpha-ketoglutaric acid and PBS every day after myocardial infarction, and after 2 weeks of continuous injection, detecting the cardiac function (EF%, FS%, LVIDs and LVIDd at 4 th week after operation) by two-dimensional echocardiography; masson staining for myocardial infarction area (4 weeks post-surgery); immunofluorescent staining detected the expression of proliferation markers (EdU pulse labeling). The results show that α -ketoglutarate improves cardiac function (fig. 7), reduces infarct size (fig. 8), and promotes adult cardiomyocyte proliferation (fig. 9).
Example 4
Alpha-ketoglutarate regulates and controls H3K27me3 in myocardial cells by activating JMJD3, and further promotes proliferation of myocardial cells
(1) Comparing the expression level of H3K27me3 in mouse myocardium in proliferation state and in non-proliferation state and the effect of alpha-ketoglutarate on H3K27me3 expression.
Newborn and adult mice were selected and divided into a control injection group (PBS) and an α -ketoglutaric acid injection group. Nucleoprotein in the myocardial tissue was extracted using a nucleoprotein extraction kit (beyond) and the expression level of H3K27me3 in the myocardial tissue was detected using Western blot. The results show that the expression level of H3K27me3 in the heart of the adult non-proliferative mouse is obviously increased, and the expression level of H3K27me3 can be reversed by alpha-ketoglutaric acid injection (figure 10).
(2) Alpha-ketoglutarate promotes myocardial proliferation by regulating JMJD3
Extracting the newborn cardiac muscle cells (the method is the same as the above): after siRNA interfered JMJD3, the influence of alpha-ketoglutarate on the proliferation capacity of the myocardial cells of the newborn mice was observed. The experimental groups were Scramble group, siRNA group, Scramble + α -ketoglutarate group, siRNA + α -ketoglutarate group. Quantification of cardiomyocyte proliferation: immunofluorescent staining detected cell proliferation markers (PH 3). The results suggest that the cardiomyocyte proliferation-promoting effect of ketoglutarate was counteracted by knock-down of JMJD3 (fig. 11).
(3) Alpha-ketoglutarate can relieve H3K27me3 inhibition of cell cycle regulatory gene by activating JMJD3 to promote proliferation of myocardial cells
Respectively treating 24 hours of cells with alpha-ketoglutaric acid and PBS, cracking the cells, performing co-immunoprecipitation with an H3K27me3 antibody (anti-H3K27me3), collecting DNA, performing high-throughput sequencing, and utilizing GO analysis and KEGG analysis to study the relation between the H3K27me3 treated with alpha-ketoglutaric acid and the biological processes of cell proliferation and division. As shown in fig. 12, alpha-ketoglutarate treatment reduced the enrichment of H3K27me3 at the transcription start site; furthermore, cell cycle-associated gene analysis found that α -ketoglutarate reduced the enrichment of H3K27me3 on ccnd1, cdk1, cdk4, cdk6 (fig. 13).
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (5)
1. Application of alpha-ketoglutaric acid in preparing medicine for treating myocardial infarction is provided.
2. The use according to claim 1, wherein the treatment is in particular:
a. repairing cardiac function damage caused by myocardial infarction;
b. reducing scar area caused by myocardial infarction;
c. promoting proliferation of myocardial cells.
3. The use of claim 2, wherein the cardiac function comprises left ventricular ejection fraction, left ventricular short axis shortening rate, left ventricular end systolic volume, and left ventricular end diastolic volume.
4. The use according to any one of claims 1 to 3, wherein the mechanism of α -ketoglutarate in the treatment of myocardial infarction is: through activating the activity of JMJD3, the inhibition of H3K27me3 in the cardiac muscle cells on the cell cycle is reduced, and the proliferation of the cardiac muscle cells is promoted.
5. Use of a pharmaceutical composition for treating myocardial infarction, said pharmaceutical composition comprising α -ketoglutarate of claim 1 and one or more pharmaceutically acceptable carriers or excipients.
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