CN114306334A - Application of songorine in adriamycin cardiotoxicity resistant medicine - Google Patents
Application of songorine in adriamycin cardiotoxicity resistant medicine Download PDFInfo
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Abstract
The invention discloses an application of songorine in an adriamycin cardiotoxicity resistant medicament, which comprises an application in medicaments with adriamycin cardiotoxicity resistance, heart failure resistance, cardiac ejection fraction increase and myocardial mitochondria protection functions. The songorine is applied to the medicines for resisting adriamycin cardiotoxicity and heart failure, so that the heart ejection fraction is effectively enhanced, the myocardial apoptosis is prevented, the myocardial mitochondrial function is protected, the myocardial fibrosis is reduced, the adriamycin is not reduced to inhibit the tumor cell proliferation while the myocardial cell activity is increased, the application and treatment effect is obvious, no obvious side effect exists, and the songorine has good medicinal prospect.
Description
Technical Field
The invention belongs to the field of medical biology, and particularly relates to application of songorine in an adriamycin cardiotoxicity resistant medicament.
Background
Doxorubicin (Doxorubicin, DOX) is a highly potent, broad-spectrum anthracycline antineoplastic drug, one of the most effective core chemotherapeutic drugs in a variety of chemotherapeutic regimens currently widely used for solid tumors and hematological malignancies. However, in the clinical application process, the adriamycin hydrochloride has strong damage effect on myocardial cells, generates adriamycin cardiotoxicity, and severely restricts the clinical application of the adriamycin. Dexrazoxane (DEX) is the only drug approved by the FDA in the united states for the prevention of doxorubicin cardiotoxicity, but exacerbates the myelosuppression caused by chemotherapeutic drugs because it may reduce the anti-tumor effect of anthracyclines. There is still a big debate on the clinical application strategy of dexrazoxane. Therefore, the search for safe and effective anti-adriamycin cardiotoxicity medicaments has important clinical application value.
Songcoline (Songorine) is a C-20 type aconite alkaloid, is one of the main chemical components of the traditional Chinese medicine aconite, has obvious pharmacological action and good clinical application prospect, and has been reported to have obvious arrhythmia, antianxiety, anti-inflammatory and insecticidal activity. Chinese patent publication No. CN113413383A discloses the use of songgaoling in the preparation of a medicament for treating arthritis. However, the application of songorine in the adriamycin cardiotoxicity resisting medicine is not reported at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of songgaoling in the anti-adriamycin cardiotoxicity medicine aiming at the defects of the prior art. The songorine is applied to the medicines for resisting adriamycin cardiotoxicity and heart failure, effectively enhances the cardiac ejection fraction, resists myocardial cell apoptosis, protects the myocardial mitochondrial function, reduces myocardial fibrosis, does not reduce the effect of adriamycin on inhibiting tumor cell proliferation while increasing the myocardial cell activity, has obvious application and treatment effects, has no obvious side effect, and has good medicinal prospect.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: the application of songgoline in adriamycin cardiotoxicity resistant medicines is characterized in that the structural formula of songgoline is as follows:
the application of the songgoline in the adriamycin cardiotoxicity resistant medicine is characterized in that the songgoline is an oral preparation.
The application of the songorine in the adriamycin cardiotoxicity resisting medicines is characterized by comprising the application in the medicines with the adriamycin cardiotoxicity resisting, heart failure resisting, cardiac ejection fraction increasing and myocardial mitochondria protecting functions.
Compared with the prior art, the invention has the following advantages:
1. the songorine provided by the invention is applied to the medicines for resisting adriamycin cardiotoxicity and heart failure, effectively enhances the cardiac ejection fraction, resists myocardial cell apoptosis, protects the myocardial mitochondrial function, reduces myocardial fibrosis, does not reduce the tumor cell proliferation inhibition effect of adriamycin while increasing the myocardial cell activity, has obvious application and treatment effects, has no obvious side effect, and has good medicinal prospects.
2. The songguoling is applied to the adriamycin cardiotoxicity resistant medicine in the form of an oral preparation, is simple and convenient and is easy to realize.
3. The application dosage of the songgaoling to clinical patients is 0.5 mg/kg-1.0 mg/kg.
The technical solution of the present invention is further described in detail by the accompanying drawings and examples.
Drawings
FIG. 1 is a graph of the results of the Songcoline antagonism doxorubicin cardiotoxic cell viability of the present invention.
FIG. 2 is a graph showing the result of the mitochondrial membrane potential of cardiomyocytes according to the present invention.
FIG. 3 is a graph showing the results of the cell viability of Songolin of the present invention on K562 cells resistant to doxorubicin.
FIG. 4a is a graph showing the results of cardiac ultrasound in mice of the present invention.
FIG. 4b is a graph showing the results of the left ventricular short axis shortening rate of the mouse of the present invention.
FIG. 4c is a graph of left ventricular ejection fraction results for mice of the present invention.
FIG. 5 is a graph showing the results of CK, CK-MB and LDH activities in the sera of mice of the present invention.
FIG. 6 shows the results of staining heart tissues of mice in each group of the present invention.
FIG. 7 is a graph showing the results of Masson's staining of cardiac tissue in each mouse group according to the present invention.
FIG. 8 is a graph of statistical analysis of the fractional collagen volume in heart tissue of mice in accordance with the present invention.
FIG. 9 is a transmission electron microscope image of the mouse myocardial tissue of the present invention.
Detailed Description
The invention provides a detailed description of the application of songorine in adriamycin cardiotoxicity resistant medicines.
Cellular level evaluation of anti-doxorubicin cardiotoxicity effect of songgaoling
1. Evaluation of cell viability
H9c2 cardiomyocytes at 5X 103The cells were seeded in 96-well plates at a density of one well and then at 37 ℃ with a 5% by volume content of CO2Culturing in a constant temperature incubator until the cell density is about 70-80%, discarding the culture solution, adding culture solution containing Songholing with different concentrations including 1 μ M, 10 μ M, 50 μ M and 100 μ M, and directly adding culture solution containing 2 μ M adriamycin after 6h to obtain sample group; according to the preparation process of the sample group, an adriamycin model group (2 μ M adriamycin), a normal control group (cells, no adriamycin) and a blank control group (cells, no adriamycin) were additionally set. Culturing the above groups for 18h, discarding the culture solution, adding 110 μ L CCK8 working solution (10 μ L CCK8 in 100 μ M DMEM medium) into each well, incubating for 3h, and detecting absorbance A at 450nm450And calculating the cell viability by taking the blank control group as a reference, wherein the calculation formula is as follows:
fig. 1 is a graph showing the results of the inventive songorine antagonizing doxorubicin cardiotoxic cell activity, and it can be seen from fig. 1 that, compared with the normal control group, the doxorubicin model group, i.e., DOX group, has cell activity of 50% to 60%, indicating that doxorubicin is toxic to cells, while when the songorine sample group with different concentrations is added, cell activity is increased along with the increase of songorine drug concentration, showing that songorine has the effect of inhibiting doxorubicin-induced myocardial damage.
2. Measurement of myocardial cell mitochondrial membrane potential
H9c2 cardiomyocytes at 8X 104The density of each well was seeded on 6-well plates and then the CO content was 5% by volume at 37 ℃2Culturing in a constant temperature incubator until the cell density is about 70-80%, removing the culture medium by suction, adding a culture solution containing 25 mu g/mL songguoling, and directly adding a culture solution containing 1 mu M adriamycin after 6 hours; after continuing culturing for 18h, washing with PBS for 1 time, adding fresh 1mL of complete culture medium (DMEM medium) and 1mL of mitochondrial membrane potential fluorescent dye JC-1 staining working solution into each well, fully mixing, and incubating for 20min at 37 ℃; next, an appropriate amount of JC-1 staining buffer (1X) was prepared in a ratio of 1mL of JC-1 staining buffer (5X) to 4mL of ultrapure water, and the mixture was placed in an ice bath. After incubation at 37 ℃, the supernatant was discarded and washed 2 times with JC-1 staining buffer (1 ×), maintaining the ice bath effect at 4 ℃ better, and 2mL complete medium was added to each well to prepare a sample group. According to the preparation process of the sample group, an adriamycin model group (2 μ M adriamycin) and a normal control group (with cells and without adriamycin) are additionally set. The sample group, the doxorubicin model group and the normal control group were observed under a fluorescence microscope, respectively, and the results are shown in fig. 2.
Fig. 2 is a graph showing the result of mitochondrial membrane potential of cardiomyocytes according to the present invention, where green fluorescence appears to indicate that the mitochondrial membrane potential is decreased, and the cells are likely to be in an early stage of apoptosis, and red fluorescence appears to indicate that the mitochondrial membrane potential is normal, and the state of the cells is also normal, as can be seen from fig. 2, most of the normal control group is red fluorescence, and contains less green fluorescence, indicating that the cells in the normal control group grow well, and compared with the normal control group, the green fluorescence of the doxorubicin model group, i.e., DOX group, is significantly increased, indicating that doxorubicin produces toxicity to the cells, so as to cause apoptosis, whereas compared with the doxorubicin model group, the green fluorescence in the sample group is decreased, indicating that songorine effectively inhibits the toxicity of doxorubicin to the cardiomyocytes, and reduces the apoptosis of the cardiomyocytes.
Effect of Songcoline on antitumor Activity of Adriamycin
Taking human chronic leukemia cell K562 with good growth state at a ratio of 1 × 104The density per well (50. mu.L) was inoculated into a 96-well cell culture plate, and culture solutions (50. mu.L each) containing Songoline (Songoline) at different concentrations including 5. mu.M, 15. mu.M, 25. mu.M, 50. mu.M, and 100. mu.M were added thereto after 6 hours, respectively, culture solutions containing 9. mu.M, 5. mu.M, and 0. mu.M Doxorubicin (DOX) were added to obtain a drug-treated group; according to the preparation process of the drug treatment, a normal control group (with cells and without adriamycin) and a blank control group (without cells and without adriamycin) are additionally arranged, 10 mu L of CCK8 solution is added into each group after 18h for incubation for 2h, and the absorbance value A is detected at 450nm450And calculating the inhibition rate by taking the blank control group as a reference, wherein the calculation formula is as follows:
fig. 3 is a result graph of cell viability of songorine of the present invention against K562 cells with doxorubicin, as shown in fig. 3, in this experiment songorine did not inhibit the proliferation of K562 cells and did not affect the inhibition of K562 cells by doxorubicin when used in combination with doxorubicin.
Animal level evaluation of anti-doxorubicin cardiotoxicity effect of Songcoline
1. Mouse adriamycin myocardial injury model establishment and grouping
6-8 week-old SPF-grade C57BL/6 male mice were purchased from the laboratory animal center of air force military medical university and were fed in the animal rooms of the traditional Chinese medicine and natural pharmacology research laboratory of air force military medical university. After 7 days of adaptive feeding and observation, healthy and active mice were selected for experiments. The selected mice were randomly divided into 4 groups: the Control group (Control) comprises 10 animals, the model group comprises 20 animals of adriamycin (DOX), 18 animals of Sonogoling low-dose (Songoline-L) and 19 animals of Sonogoling high-dose (Songoline-H). The control mice were injected with 0.2ml/20g of normal saline in the abdominal cavity 1 time a week for 4 times; and injecting 6mg/kg of adriamycin into the abdominal cavity of the model group mice 1 time per week for 4 weeks, wherein the accumulated dose reaches 24mg/kg, so as to establish an adriamycin myocardial injury model, wherein the mice in the control group and the model group are intragastrically injected with normal saline every day, and the mice in the Sonogoling low dose group and the Sonogoling high dose group are intragastrically administered with 5mg/kg and 10mg/kg of Sonogoling solution respectively every day while injecting the same amount of adriamycin every week.
2. Effect of mouse survival and Heart/body weight ratio
After the control group and the model group were continuously administered for 4 weeks, it was found that the model group mice exhibited symptoms such as lassitude, sleepiness, curling, lusterless hair, and decreased activity, and after the experiment, the weight and heart weight of each mouse in each group were recorded and treated accordingly, and the results are shown in table 1.
TABLE 1 mouse mortality, Heart weight, weight changes, organ coefficients Table
Note: in Table 1, "#" indicates that p <0.05(n >6) in the doxorubicin group compared with the control group; ") indicates that p <0.05(n >6 in the low dose or high dose treatment with sonoguang versus normal groups.
As can be seen from table 1, the mortality of the mice in the doxorubicin group was 30% compared with the control group, while the mortality of the mice in the songgoline low dose group and the songgoline high dose group was respectively reduced by 7.78% and 14.21% compared with the doxorubicin group, the heart/body weight of the mice in the doxorubicin group was statistically significant (p <0.05) compared with the control group, and the reduction of the body weight of the mice in the songgoline low dose group and the songgoline high dose group was significantly reduced by the co-treatment with DOX and songgoline compared with the doxorubicin group, which indicated that songgoline effectively reduced the mortality and reduced the weight reduction of the mice with doxorubicin-induced myocardial damage.
3. Influence of cardiac function index
After the mice in the control group and the model group are continuously acted for 4 weeks, uniformly smearing a proper amount of depilatory cream on the heart parts of the mice by using a cotton swab, and then carefully wiping the mice with a wet tissue to depilate to completely expose the skin of the heart parts; anaesthetizing a mouse with 1.0% isoflurane for 5min, fixing the mouse on a constant-temperature operation board at 37 ℃ with the chest facing upwards, uniformly smearing a proper amount of special coupling agent for detection on the heart, placing a matched probe of a Vevo 2100 high-resolution small animal ultrasonic imaging system on the chest of the mouse, observing images, adjusting the proper position, observing a long-axis section of a sternum through B-type ultrasound, storing the images, switching to M-type ultrasound, and storing the image at the position with the maximum diameter of the lower left ventricle, wherein the result is shown in figure 4 a; meanwhile, Left Ventricular Ejection Fraction (LVEF) and Left Ventricular short axis Shortening (LVFS) were obtained by processing with the Vevo 3.1.1 software, and the results are shown in fig. 4b and 4 c.
Fig. 4a is a cardiac ultrasound result graph of a mouse of the present invention, fig. 4b is a left ventricular short axis shortening rate result graph of a mouse of the present invention, fig. 4c is a left ventricular ejection fraction result graph of a mouse of the present invention, and as can be seen from fig. 4a to fig. 4c, cardiac function indexes LVFS and LVEF of a mouse of an adriamycin group are both significantly reduced (p <0.01) compared with a control group; compared with the adriamycin group, after the low-dose group of the songguang and the high-dose group of the songguang are jointly treated by the combination of DOX and the songguang, the cardiac function index of the mice is remarkably improved (p is less than 0.05), and the cardiac function index of the mice in the high-dose group of the songguang is higher than that in the low-dose group of the songguang, which indicates that the heart function damage of the mice induced by DOX can be improved by the treatment of the songguang, and the improvement effect of the high-dose group of the songguang is better than that in the low-dose group of the songguang.
4. Detection of CK, CK-MB and LDH activity of serum
After the control group and the model group were continuously administered for 4 weeks, blood was collected from the eyes of each group of mice, and serum was collected by centrifugation to detect Creatine Kinase (CK), creatine kinase isoenzyme (CK-MB), and Lactate Dehydrogenase (LDH), and the results are shown in FIG. 5.
FIG. 5 is a graph showing the results of CK, CK-MB and LDH activities in the sera of mice of the present invention, and it can be seen from FIG. 5 that CK, CK-MB and LDH activities in the sera of the mice of the doxorubicin group are significantly increased (p <0.01) compared with the control group; compared with the adriamycin group, the LDH activity of the mice subjected to intragastric administration of the songorine including the high-dose group and the low-dose group of the songorine is remarkably reduced (the p values are both less than 0.05).
5. HE staining for myocardial damage
Taking the whole heart of each group of mice from the control group and the model group, fixing by adopting 4% paraformaldehyde solution, dehydrating, clearing tissues, embedding paraffin, slicing, dewaxing, staining by hematoxylin-eosin (H-E), dehydrating and sealing; then, the morphology of the myocardial tissue was observed under a light microscope, photographed, and analyzed by contrast, and the results are shown in FIG. 6.
Fig. 6 shows the staining results of the heart tissues of the mice in each group of the invention, wherein a in fig. 6 is a control group, B is an adriamycin group, C is a songgoling low dose group, and D is a songgoling high dose group, and it can be seen from fig. 6 that the myocardial tissue cells of the mice in the control group are regularly and regularly arranged, the cells are short column-shaped, and the myocardial texture is clear; the myocardial cell damage phenomenon in the myocardial tissues of rats in the adriamycin group, namely DOX group is serious, the arrangement of myocardial fibers is disordered, a plurality of parts are broken, the arrangement of cell nuclei is irregular and even lost, the myocardial cells are vacuolated, the cytoplasm is subjected to particle degeneration, and inflammatory cell infiltration can be seen; after the treatment of the Songcoline in the high-dose group and the low-dose group, the arrangement of the myocardial tissue cells tends to be tidy gradually, the damage degree of the myocardial cells is obviously improved, vacuole and inflammatory phenomena are reduced gradually, and the cell textures are clear gradually.
6. Masson staining for myocardial damage
1/4 myocardial tissues of the apex of the left ventricle of the heart of each mouse group are taken from the control group and the model group, fixed by 4 percent paraformaldehyde solution, dehydrated, transparent, embedded, sliced and stained by massson. The morphology of the myocardial tissue was observed under a light microscope and photographed, and the fibrosis area was evaluated by calculation using a Collagen Volume Fraction (CVF), and the results of comparative analysis were shown in fig. 7 and 8.
Fig. 7 is a staining result graph of Masson of cardiac tissue of each group of mice of the present invention, in fig. 7, a is a control group, B is an doxorubicin group, C is a songgoline low dose group, D is a songgoline high dose group, collagen fibers in the graph are blue, cardiomyocytes are red, and it can be seen from fig. 7 that the doxorubicin group, i.e., DOX myocardial fibrosis area is significantly increased compared to the normal group; the Songcoline treatment group comprises a Songcoline high-dose group and a Songcoline low-dose group, and the myocardial interstitial fibrosis of the mice is obviously reduced compared with that of the DOX group, and the fibrosis of the Songcoline high-dose group is obviously lower than that of the Songcoline low-dose group.
Fig. 8 is a statistical analysis chart of the fractional collagen volume in the heart tissue of each group of mice according to the present invention, and it can be seen from fig. 8 that the myocardial interstitial fibrosis of the doxorubicin group was significantly increased (p <0.01) compared to the control group, and the fibrosis caused by doxorubicin was alleviated by the songroling-treated group including the songroling high dose group and the songroling low dose group.
7. Ultrastructural analysis of myocardial tissue
For the above control group and model group, 1cm of cardiac apex part of mouse heart was taken3Myocardial tissue was fixed in a fixative (2.5% glutaraldehyde and 1% osmic acid), observed by transmission electron microscopy and photographed, and analyzed by contrast, and the results are shown in FIG. 9.
Fig. 9 is a transmission electron microscope image of the mouse cardiac muscle tissue of the invention, in fig. 9, a is a control group, B is an adriamycin group, C is a songguoling low dose group, and D is a songguoling high dose group, and it can be seen from fig. 9 that the cardiac muscle filaments of the control group mouse are arranged neatly, the shape of mitochondria is intact, the distribution and size are uniform, and few vacuole-like changes exist; the myocardial muscle filaments of the model group are disorderly arranged, the muscle filaments are broken, mitochondria are obviously swollen, a large amount of vacuoles are changed, partial mitochondrial cristae is reduced or even disappears, and the structure is incomplete and fuzzy, wherein the arrangement of the myocardial muscle filaments of the Songolin treatment group including the Songolin high-dose group and the Songolin low-dose group is relatively regular, the mitochondria are obviously repaired compared with the model group, the mitochondrial cristae number is improved, the structure is relatively complete, and the mitochondria are only slightly swollen.
In conclusion, the in vitro cell viability test and the in vivo animal experiment show that the songorine increases the myocardial cell viability, does not reduce the tumor cell proliferation inhibition effect of adriamycin, enhances the cardiac ejection fraction, resists myocardial cell apoptosis, protects the myocardial mitochondrial function, reduces myocardial fibrosis, has obvious treatment effect in the application of medicines with adriamycin cardiotoxicity resistance and heart failure resistance, has no obvious side effect, and has good medicinal prospect.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.
Claims (3)
1. The application of songgoline in adriamycin cardiotoxicity resistant medicines is characterized in that the structural formula of songgoline is as follows:
2. the use of songorine according to claim 1 in an anti-doxorubicin cardiotoxicity medicament, wherein the songorine is an oral formulation.
3. The use of songorine according to claim 1 in anti-doxorubicin cardiotoxicity medicaments, comprising the use in medicaments with anti-doxorubicin cardiotoxicity, anti-heart failure, increasing cardiac ejection fraction, protecting myocardial mitochondrial function.
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| CN115919850A (en) * | 2022-12-01 | 2023-04-07 | 中国人民解放军空军军医大学 | Anti-adriamycin cardiotoxicity traditional Chinese medicine monomer composition and preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115919850A (en) * | 2022-12-01 | 2023-04-07 | 中国人民解放军空军军医大学 | Anti-adriamycin cardiotoxicity traditional Chinese medicine monomer composition and preparation method and application thereof |
| CN115919850B (en) * | 2022-12-01 | 2024-03-19 | 中国人民解放军空军军医大学 | Traditional Chinese medicine monomer composition for resisting doxorubicin cardiotoxicity and preparation method and application thereof |
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