CN109528753B - Application of oligomeric hyaluronic acid or salt thereof in preparation of medicine for treating myocardial infarction - Google Patents
Application of oligomeric hyaluronic acid or salt thereof in preparation of medicine for treating myocardial infarction Download PDFInfo
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- CN109528753B CN109528753B CN201811564130.2A CN201811564130A CN109528753B CN 109528753 B CN109528753 B CN 109528753B CN 201811564130 A CN201811564130 A CN 201811564130A CN 109528753 B CN109528753 B CN 109528753B
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- hyaluronic acid
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- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
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- 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
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Abstract
The invention discloses application of oligomeric hyaluronic acid or a salt thereof in preparing a medicament for treating myocardial infarction. The oligomeric hyaluronic acid and the salt thereof can effectively treat myocardial infarction, have obvious curative effect, provide a new medicament for clinically treating myocardial infarction, and have good clinical application prospect.
Description
Technical Field
The invention relates to application of oligomeric hyaluronic acid or a salt thereof in preparing a medicament for treating myocardial infarction.
Background
Myocardial infarction is also called myocardial infarction and refers to myocardial necrosis caused by acute and persistent ischemia and hypoxia (coronary insufficiency); on the basis of coronary artery pathological changes, the blood of coronary arteries is interrupted, so that severe and persistent acute ischemia of corresponding cardiac muscle occurs, and finally ischemic necrosis of the cardiac muscle is caused; acute myocardial infarction patients have persistent acute pain after sternum, fever, increased white blood cell count, increased serum myocardial enzyme and a series of characteristic evolution of acute myocardial injury, ischemia and necrosis reflected by electrocardiogram, can have arrhythmia, shock and heart failure, belong to the serious type of coronary heart disease, can be complicated by arrhythmia, shock or heart failure and other complications, and can be threatened to life. The disease is most common in europe and the united states, and about 150 million people suffer myocardial infarction in the united states each year. China has a remarkable rising trend in recent years, newly issues at least 50 ten thousand every year, and finds out at least 200 ten thousand.
No report of oligomeric hyaluronic acid and salt thereof for treating myocardial infarction is found.
Disclosure of Invention
The technical scheme of the invention provides new application of oligomeric hyaluronic acid and salt thereof.
The invention provides application of oligomeric hyaluronic acid or a salt thereof in preparing a medicament for treating myocardial infarction and/or promoting myocardial regeneration.
Preferably, the oligomeric hyaluronic acid is a hyaluronic acid with a molecular weight of less than 5000 Da.
Preferably, the oligomeric hyaluronic acid is hyaluronic acid with a number average molecular weight of 4750 Da.
Preferably, the oligomeric hyaluronic acid is prepared according to the following method: taking hyaluronic acid, performing enzymolysis and dialysis, collecting parts with molecular weight of 1000-5000 Da, and freeze-drying.
Preferably, the method for enzymolysis is as follows: dissolving hyaluronic acid per 100mg in 50mL of 0.1M sodium acetate buffer, adding bovine testis hyaluronidase with 20000 activity units, incubating at 37 deg.C for 8h, adding trifluoroacetic acid to neutralize hyaluronidase, centrifuging, and collecting supernatant; the hyaluronic acid is preferably hyaluronic acid having a molecular weight of 100 ten thousand.
Preferably, the medicament is a preparation prepared by taking an effective amount of oligomeric hyaluronic acid and/or salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
Preferably, the formulation is an injection.
Preferably, the medicament for promoting myocardial regeneration is a medicament for reducing myocardial cell apoptosis, increasing the number of myocardial blood vessels and promoting myocardial cell proliferation.
The invention also provides a medicament for treating myocardial infarction, which is a preparation prepared by taking effective amount of oligomeric hyaluronic acid and/or salt thereof as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
Preferably, the oligomeric hyaluronic acid is a hyaluronic acid with a molecular weight of less than 5000 Da.
Preferably, the oligomeric hyaluronic acid is hyaluronic acid with a number average molecular weight of 4750 Da.
Preferably, the formulation is an injection.
The oligomeric hyaluronic acid and the salt thereof can effectively treat myocardial infarction, have obvious curative effect, can reduce the apoptosis of myocardial cells, increase the number of myocardial blood vessels, promote the survival of left ventricular cardiomyocytes, can promote the macrophages to secrete growth factors VEGF by regulating and controlling the typing of the macrophages, promote the proliferation of the myocardial cells, promote the regeneration of the myocardium, further prevent heart failure and improve the curative effect of heart disease treatment, and have good clinical application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 characterization of o-HA
FIG. 2 evaluation of the therapeutic effect of o-HA on mice.
FIG. 3 assay of cTn I release from mouse serum after MI
FIG. 4 histopathological analysis of myocardial tissue. A, TUNEL; b, CD 31; c, Ki67 expression level.
FIG. 5 pathological HE staining of each major organ after the end of treatment of mice in different experimental groups.
FIG. 6 shows the effect of different experimental groups on macrophage migration and invasion.
FIG. 7 flow detection of macrophage typing and WB detection of VEGF expression by different experimental groups
Detailed Description
EXAMPLE 1 preparation of the oligomeric hyaluronic acid of the present invention
100mg of h-HA of 100 ten thousand molecular weight was dissolved in 50mL of 0.1M sodium acetate buffer (pH 5.4), 20000 units of bovine testicular hyaluronidase in active units was added, and incubated at 37 ℃ for 8 h. 10mL of the solution was taken out, and 1mL of 100% trifluoroacetic acid (TCA) was added to neutralize hyaluronidase. The resulting enzyme precipitate was removed by centrifugation. Collecting supernatant, dialyzing in dialysis bag with molecular weight of 1000Da at 4 deg.C for 72 hr, and changing water at least 6 times. Collecting the liquid in the dialysis bag, and continuously dialyzing for 72h at 4 ℃ in the dialysis bag with the molecular weight of 5000 Da. After dialysis, the external aqueous phase was collected and lyophilized, and the lyophilized o-HA sample was stored at 4 ℃ until use.
The molecular weight distribution and homogeneity measurements of the product will be determined by GPC analysis.
As a result, as shown in FIG. 1, the o-HA was prepared by enzymatically degrading h-HA. As can be seen in FIG. 1, the number average molecular weight of o-HA was 4750Da and the PDI was lower than 1.52, indicating that the molecular weight distribution of o-HA oligosaccharides was uniform, thereby confirming the success of o-HA production.
Test example 1 therapeutic action of sodium oligohyaluronate on myocardial infarction and myocardial ischemia
1. Experimental Material
Oligomeric sodium hyaluronate, prepared according to the method of example 1.
2. Experimental methods
MI modeling surgical procedure
Male C57 mice 6-8 weeks old were used as our model building subjects and injected intraperitoneally with ketamine (100 mg/kg)-1) And xylazine (10 mg/kg)-1) Mice were anesthetized, thoracotomy was performed using a small animal ventilator, and the left anterior descending coronary artery was ligated. After ligation, the wound was closed and the mice were ventilated for 30 minutes until the mice lungs re-expanded. The sham group was also operated in the same manner except that the left anterior descending branch was not ligated.
The experiment was divided into 4 groups, Sham, PBS, h-HA and o-HA, 8 mice per group. The administration mode is intravenous injection, and the injection dosage of h-HA and o-HA is 5 mg/kg. After 28 days of treatment, mice were sacrificed and heart, liver, spleen, lung, and kidney were collected for follow-up experiments, a portion was fixed in 4% formalin solution for HE and MASSON pathology analysis (n ═ 4); another portion was frozen sections for TUNEL, Ki67 and CD31 immunohistochemical analysis (n-4).
2.2.MI biomarkers following myocardial ischemic injury in mice, serum (n ═ 6) was collected by orbital bleeds at various time points, 6, 8, 12, 24, 28, 72, 96, 120, 144, 168h, and the concentrations of cTn I in the sera of mice from different treatment groups were measured by ELISA kits.
2.3. Left ventricular myocardial pathology analysis 28 days after treatment of mice, hearts of mice of each experimental group were collected, a portion was fixed in 4% formalin, paraffin sections were stained by HE and MASSON, and myocardial ischemia degree was analyzed.
Another part of the mouse, which was a fresh heart, was cut into tissue pieces with a thickness of 3mm, stained with TTC, and photographed by a digital camera to analyze the myocardial ischemia area.
2.4. Mouse cardiac myocardial regeneration assay
The collected heart tissue of the remaining part was analyzed by frozen section immunohistochemistry for various detection indicators including TUNEL, Ki67 and CD31, and myocardial apoptosis, neovascularization and proliferation of myocardial cells in the myocardial tissue were analyzed.
2.5. Safety and toxicity evaluation 28 days after treatment, the collected major organs of liver, spleen, lung and kidney were fixed in 4% formalin, and HE-stained by paraffin section for evaluation of o-HA toxicity. In addition, blood biochemical analysis was performed on the collected mouse blood.
Study of the mechanism of o-HA on myocardial regeneration
The influence of o-HA on the induction of macrophage migration and invasion and macrophage polarization is analyzed through migration, invasion, flow and protein experiments.
3. Results of the experiment
3.1 o-HA reduction of myocardial infarct size
FIG. 2A shows that, after TTC staining of left ventricular myocardial tissue, a larger white area indicates a larger myocardial infarction area, which is reduced by about 90% after o-HA treatment compared to NS and h-HA groups. The results show that o-HA significantly reduces myocardial fibrosis area and can effectively treat MI. HE staining showed that (2C), the o-HA treated group had a significantly reduced degree of cardiomyocyte necrosis compared to the other treated groups, and there was no significant inflammatory cell infiltration within the myocardial tissue. Fig. 2D shows that MASSON staining of the paraffin sections of the heart shows that the blue occupied area reflects the degree of left ventricular fibrosis, indicating that the degree of left ventricular myocardial fibrosis in the o-HA treatment group is significantly reduced and the myocardial infarction area is significantly reduced compared to other experimental groups.
The experimental result shows that the oligomeric hyaluronic acid can effectively treat myocardial infarction, and the effect is obviously superior to that of the hyaluronic acid with the common molecular weight.
3.2MI biomarker detection
cTn I is a specific marker produced after cardiac muscle cell death by MI, and its content in serum reflects the irreversible death degree of cardiac muscle cells. As shown in FIG. 3, the concentrations of cTn I in serum at 24h in the NS group and the h-HA group were 598.2. + -. 37.1and 489.5. + -. 8.2Pg/mL, respectively, and then decreased and continued at higher concentrations for up to 28 days. In the o-HA group, cTn I concentration at 24h was lower than that in both groups, 378. + -. 12.5Pg/mL, and at a concentration level as low as that in the Sham group up to 28 days.
The experimental result shows that the o-HA can effectively protect and reduce the death of the myocardial cells
3.3 myocardial apoptosis and regeneration assays
FIG. 4A shows that we used TUNEL staining analysis to significantly reduce the proportion of TUNEL-positive cardiomyocytes in the o-HA treatment group; as shown in fig. 4B and 4C, the proportion of CD 31-positive cells in the o-HA-treated myocardial tissue was significantly increased compared to the other groups, and Ki 67-positive myocardial cells in the myocardial tissue were significantly increased compared to the other treated groups. The result shows that the o-HA can effectively protect the myocardial cells, reduce the apoptosis of the myocardial cells, increase the angiogenesis of the myocardial tissues and increase the proliferation of the myocardial cells.
3.4 evaluation of safety and toxicity
Pathological observation of HE stained tissue after the end of treatment showed no pathological changes in the small major organs and no infiltration of inflammatory cells after o-HA treatment (fig. 5). The results show that the o-HA HAs high safety and low toxic and side effects when used for MI treatment.
3.5 mechanism study of macrophage by o-HA
Through scratch and Transwell experiments, we found that o-HA was able to promote migration and invasion of macrophages (fig. 6); flow cytometry analysis revealed that o-HA was able to promote macrophage polarization to M2 type and promote vascular endothelial growth factor VEGF expression (fig. 7). We believe this will be the main reason for o-HA to promote myocardial regeneration.
4 conclusion
The experiment of the invention proves that the oligomeric hyaluronic acid o-HA can effectively treat myocardial infarction, and the effect is obviously superior to the hyaluronic acid h-HA with common molecular weight.
In addition, the experiment of the invention also proves that the oligomeric hyaluronic acid o-HA can reduce the apoptosis of cardiac muscle cells, increase the number of cardiac muscle blood vessels, promote the survival of cardiac muscle cells of the left ventricle, can regulate and control the typing of macrophages, promote the macrophages to secrete growth factor VEGF, promote the proliferation of cardiac muscle cells, and HAs the effect obviously superior to that of the hyaluronic acid h-HA with the common molecular weight.
In conclusion, the oligomeric hyaluronic acid and the salt thereof can effectively treat myocardial infarction, have remarkable curative effect and small side effect, provide a new medicament for clinically treating myocardial infarction, and have good clinical application prospect.
Claims (4)
1. The application of the oligomeric hyaluronic acid or the salt thereof in preparing the medicine for treating myocardial infarction is characterized in that: the oligomeric hyaluronic acid is oligomeric hyaluronic acid with the number average molecular weight of 4750Da, and the oligomeric hyaluronic acid is prepared by the following method: taking hyaluronic acid, performing enzymolysis and dialysis, collecting part with molecular weight of 1000-5000 Da, and freeze-drying; the enzyme is bovine testicular hyaluronidase in 20000 activity units.
2. Use according to claim 1, characterized in that: the enzymolysis method comprises the following steps: dissolving hyaluronic acid per 100mg in 50mL of 0.1M sodium acetate buffer, adding bovine testis hyaluronidase with 20000 activity units, incubating at 37 deg.C for 8h, adding trifluoroacetic acid to neutralize hyaluronidase, centrifuging, and collecting supernatant; the hyaluronic acid is hyaluronic acid with a molecular weight of 100 ten thousand.
3. Use according to claim 1 or 2, characterized in that: the medicament is a preparation prepared by taking an effective amount of oligomeric hyaluronic acid and/or salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
4. Use according to claim 3, characterized in that: the preparation is injection.
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