CN111249274A

CN111249274A - Application of ginkgolide B in preparation of glioma cell activity inhibitor

Info

Publication number: CN111249274A
Application number: CN201911013105.XA
Authority: CN
Inventors: 谭舟; 王加银; 裴顺琪; 邱猛生
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2020-06-09
Anticipated expiration: 2039-10-23
Also published as: CN111249274B

Abstract

The invention relates to the field of biological medicine, aims at the problem that ginkgolide B in the prior art does not have an anti-cancer effect on malignant glioma, and discloses an application of ginkgolide B in preparation of a glioma cell activity inhibitor, wherein the glioma cell activity inhibitor comprises ginkgolide B and derivatives thereof; the glioma cells comprise one or more of glioma cell U-87MG, glioma cell U-251MG, or glioma cell A172; the glioma cell activity inhibitor is applied to a malignant brain glioma chemotherapy drug. The invention discovers that the ginkgolide B has a certain treatment effect on the malignant glioma; the ginkgolide B can inhibit the migration and proliferation of glioma cells, reduce the activity of ATP enzyme so as to influence the metabolic level of glioma cells, block the cell cycle and induce apoptosis, and provides a new alternative scheme of chemotherapeutic drugs for treating malignant glioma.

Description

Application of ginkgolide B in preparation of glioma cell activity inhibitor

Technical Field

The invention relates to the field of biological medicines, in particular to application of ginkgolide B in preparation of a glioma cell activity inhibitor.

Background

Glioblastomas (GBMs) are the most malignant of the central nervous system tumors, with a survival rate of less than 10% for more than five years in patients. Glioma grows and is characterized by invasive growth, and has no obvious boundary with normal brain tissue, so theoretically, the glioma cannot be completely excised by operation, and still has the problems of difficult treatment, poor postoperative prognosis and the like. Despite the progress of therapeutic approaches to glioblastoma over the last decade, chemotherapy is still the most mature method of treating the disease at present. However, in terms of current clinical application, the traditional chemotherapeutic drugs have the problems of great side effect, tumor drug resistance, incapability of penetrating blood brain barrier and the like. Therefore, a small molecular drug with stronger effect and less toxic and side effect on normal cells is urgently needed to treat the malignant glioma.

In recent years, biologists have studied a large number of mixtures extracted from herbs as a new source of anticancer drugs, and several natural products have been demonstrated to have significant anticancer effects against various cancers, such as leukemia, cervical cancer, ovarian cancer. The ginkgolide compound belongs to a terpenoid, consists of sesquiterpene lactone and diterpene lactone, and is an important active ingredient in ginkgo leaves. Bilobalide belongs to sesquiterpene lactone, and is the only sesquiterpene lactone compound found in ginkgo leaves at present. The ginkgolide B (BN52021) has the strongest activity and physiological activity in ginkgolide, is the strongest platelet activating factor antagonist discovered so far, can be clinically used for treating thrombus, acute pancreatitis and cardiovascular diseases, can also be used for treating metastatic cancer, has a protective effect on damaged neurons, and also has the effects of resisting oxidation and delaying aging.

The patent number CN200510109596.X is 'a pharmaceutical composition containing ginkgolide B and a preparation method and application thereof', and particularly discloses a pharmaceutical composition containing ginkgolide B, which comprises ligustrazine phosphate and ginkgolide B, wherein the weight of the ligustrazine phosphate in the pharmaceutical composition is 10-500 times that of the ginkgolide B, and the preparation method of the pharmaceutical composition.

The ginkgolide B has a certain treatment effect on metastatic cancers as a long-term medicament for treating cardiovascular and cerebrovascular diseases, but the anticancer effect of the ginkgolide B on malignant glioma is not reported in the medicament research at present.

Disclosure of Invention

The invention is in order to overcome the problem that ginkgolide B of the prior art does not have its anticancer effect on malignant glioma yet, disclose the application of ginkgolide B in preparing glioma cell activity inhibitor, find that ginkgolide B can be regarded as an active substance through the study, have certain therapeutic action on malignant glioma, the natural product, almost non-toxic, the minus effect is very little; the ginkgolide B can inhibit the migration and proliferation of glioma cells to different degrees, and can also reduce the activity of ATP enzyme so as to influence the metabolic level, block the cell cycle and induce apoptosis, thereby providing a new alternative scheme of chemotherapeutic drugs for the treatment of malignant glioma.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of bilobalide B and its derivatives in preparing glioma cell activity inhibitor is provided.

A glioma cell activity inhibitor comprises bilobalide B and derivatives thereof, wherein the bilobalide B has a specific structural formula as follows:

preferably, the glioma cells comprise one or more of glioma cell U-87MG, glioma cell U-251MG, or glioma cell A172.

The glioma cell activity inhibitor is applied to a malignant brain glioma chemotherapy drug.

With respect to the molecular mechanism of action of ginkgolide B on glioma, studies have been made to show that ginkgolide B is the strongest antagonist of Platelet Activating Factor (PAF) found so far, and data have been made to show that Platelet Activating Factor Receptor (PAFR) is specifically highly expressed in glioma, and thus ginkgolide B is highly likely to act on glioma by inhibiting PAFR. In addition, ginkgolide B can also inhibit the progression of glioma by changing Ras/MAPK signal to induce cell differentiation.

Preferably, the chemotherapeutic agent comprises bilobalide B and one or more of the pharmaceutically acceptable esters of bilobalide B.

Preferably, the administration concentration of ginkgolide B to glioma cell A172 is 1095-1100. mu.M, the administration concentration to glioma cell U-87MG is 1085-1090. mu.M, and the administration concentration to glioma cell U-251MG is 995-1000. mu.M.

Preferably, the chemotherapeutic agent comprises an inhibitor of ginkgolide B and a pharmaceutically acceptable carrier and/or excipient.

Preferably, the malignant brain glioma chemotherapeutic drug of the chemotherapeutic drug is an oral preparation or an injection preparation.

Preferably, the oral preparation is in the form of a capsule or a tablet.

Preferably, the dosage of the oral formulation is about 5-5.2mg/kg body weight, more preferably 5mg/kg body weight.

Therefore, the invention has the following beneficial effects:

(1) researches show that the ginkgolide B can be used as an active substance, has a good treatment effect on malignant glioma, is a natural product and is almost nontoxic;

(2) the ginkgolide B can obviously inhibit the migration and proliferation of glioma cells, strictly control the proliferation rate of the glioma cells and have small side effect on organisms;

(3) but also can reduce the activity of ATP enzyme to influence the metabolic level, block the cell cycle and induce apoptosis, and inhibit the formation of glioma cells from the source, so the preparation method has excellent treatment effect;

(4) provides a new alternative scheme of chemotherapeutic drugs for the treatment of malignant brain glioma and enriches the types of inhibiting drugs for malignant brain glioma.

Drawings

FIG. 1 is a graph showing the dose-effect relationship between ginkgolide B and the inhibition of cell proliferation in three glioma cell lines.

FIG. 2 is a graph showing the growth of U-87MG cells under the action of ginkgolide B.

FIG. 3 is a light microscope photograph of growth inhibition and cell morphology change of three glioma cell lines with different concentrations of ginkgolide B.

FIG. 4 is a statistical graph of the effect of ginkgolide B on cell migration distance of three glioma cells.

FIG. 5 is a bar graph (5(B)) showing the results of BrdU labeling for quantifying the effect of ginkgolide B on glioma cell proliferation (5 (a)).

FIG. 6 is a histogram (6(a)) and a statistical bar chart (6(B)) showing the effect of ginkgolide B on the growth of intracranial glioma in C57B6 mice.

Detailed Description

The invention is further described with reference to specific embodiments.

Examples

I. Effect of ginkgolide B on proliferation and toxicity of glioma cells U-87MG

Glioma cells U-87MG (purchased from ATCC) were prepared in DMEM medium containing 10% fetal bovine serum and added to a 96-well plate at 100. mu.l per well (5000 cells/well). The plates were pre-incubated in an incubator for 24 hours (37 ℃, 5% CO)₂). DMEM medium containing 10% fetal bovine serum at final concentrations of 0. mu.M, 250. mu.M, 500. mu.M, 1000. mu.M, 2000. mu.M ginkgolide B was added to wells of the plate, 200. mu.l per well, respectively. The plates were incubated in an incubator for 48 hours. The medium was discarded and replaced with DMEM medium containing 10% CCK 8. The plates were incubated in an incubator for 1 hour. The absorbance (OD value) at 450nm was measured with a microplate reader. Culture wells without glioma cells U-87MG and ginkgolide B and containing CCK8 were used as blank controls, and culture wells without ginkgolide B and containing glioma cells U-87MG and CCK8 were used as negative controls.

And (3) activity calculation: cell viability [ (%) [ a (medicated) -a (blank) ]/[ a (0 medicated) -a (blank) ] × 100

A (dosing): absorbance of wells containing glioma cells U-87MG, CCK8 and bilobalide B

A (blank): absorbance of wells with media and CCK8 without glioma cells U-87MG and bilobalide B

A (0 dosing): absorbance of wells with glioma cells U-87MG, CCK8 and no bilobalide B

Cell viability: cell proliferation Activity or cytotoxic Activity

Under the same conditions, the test was carried out using glioma cell lines A172 and U-251MG instead of U-87MG, and the results are shown in FIG. 1, wherein the half-inhibitory concentrations of ginkgolide B on glioma cell lines A172, U-87MG and U-251MG were 1098. mu.M, 1087. mu.M and 996. mu.M, respectively.

II. Inhibition of proliferation of different glioma cells by ginkgolide B

Mu.l of glioma cells U-87MG (5000 per well) were plated in 96-well plates in DMEM medium containing 10% fetal bovine serum. The plates were pre-incubated in an incubator for 24 hours (37 ℃, 5% CO)₂). DMEM medium containing 10% fetal bovine serum containing 0.5. mu.M ginkgolide B was added to the plates at 200. mu.l per well, and DMEM medium containing 10% fetal bovine serum containing an equivalent amount of PBS instead of ginkgolide B was used as a control. The plates were incubated in an incubator for an appropriate period of time (0-6 days), and the cells in the plates with and without drug added were counted and growth curves were plotted daily at fixed times.

Under the same conditions, glioma cell lines A172 and U-251MG were used instead of U-87MG respectively, and the results are shown in FIG. 2, wherein the number of cells without drug treatment is normally increased, while the proliferation speed of cells treated by ginkgolide B is extremely slow, and apoptosis occurs under high concentration of drug.

III Effect of Bilobalide B on glioma migration Capacity

1. Preparation of cell samples

A-172MG cells growing logarithmically are spread in a six-well plate, when the confluence degree of the cells reaches about 80%, a scratching experiment is carried out by using a medium-sized gun head, the cells floating are washed for 2 times by PBS, and serum-free DMEM culture media respectively containing medium-concentration (1000 mu M) and low-concentration (500 mu M) ginkgolide B and ginkgolide B are added.

2. Photographing observation

The scratch part was photographed every 24 hours under a microscope, and the scratch width was statistically analyzed.

Under the same conditions, the results of the experiment using U251 cells instead of A-172MG cells are shown in FIG. 4, and the statistical results are shown in FIG. 4 d, which shows that the migration ability of A-172MG cells treated with ginkgolide B is significantly reduced compared with that of non-drug-treated cells.

IIII and BrdU labeling method for analyzing influence of ginkgolide B on cell proliferation

1. Preparation of cell samples:

taking logarithmic growth U-87MG cells, culturing until the confluence degree is 70%, adding ginkgolide B with final concentration of 500 μ M and 1000 μ M, and culturing for 48 h.

2. BrdU labeling:

a.48 hours later BrdU (5-bromodeoxyuridine) was added at a final concentration of 90g/L and incubated at 37 ℃ for 2 hours.

B. After 2 hours incubation, the medium was discarded and the cells were washed 2 times with PBS for 5 minutes each.

3. Cell immobilization:

mu.l of cell fixative (4% paraformaldehyde) was added to each well and incubated at room temperature for 30 minutes, the fixative was discarded and washed 3 times with PBS for 5 minutes each.

4. HCl fixation:

adding 2M HCl aqueous solution to each well of the culture wells in the step 3, incubating the wells in an environment at 37 ℃ for 20 minutes, washing the wells with PBS for 2 times, adding 0.1M boric acid buffer solution to each well at pH8.5 to cover the cells, incubating the wells for 10 minutes, and washing the wells with PBS for 3 times.

5. And (3) sealing:

blocking with 5% sheep serum for 1 hour at room temperature.

6. Incubation of primary antibody and secondary antibody

After primary antibody (mouse BrdU monoclonal antibody) was incubated overnight at 4 ℃ and washed 3 times with PBS, secondary antibody (coat-anti-mouse IgG1(r1),594) was incubated for 1 hour, DAPI (4', 6-diamidino-2-phenylindole) was added 5 minutes before the end of the secondary antibody incubation in a volume ratio of 1:10000, and PBS was washed three times. Pictures were taken under a fluorescent microscope.

Under the same conditions, the results of the experiment using A172 cells instead of U-87MG cells are shown in FIG. 5, and the statistical results in FIG. 5B show that the proliferation degree of the U-87MG cells treated with ginkgolide B is significantly reduced compared with the cells not treated with drug.

7. Animal experiments

Intracranial transplantation of 5X 10C 57B6 mice at 3-4 weeks⁴～1×10⁵The GL261 cells formed brain gliomas in about 2 weeks, and the bilobalide B drug-treated group (oral gavage) and the control group were established, and the results are shown in fig. 6: the oral administration of 5mg/kg body weight of ginkgolide B was effective in inhibiting the growth of intracranial glioma, and the statistical results are shown in B of FIG. 6.

The proliferation curves in fig. 1, the cell activities of the three tumor cell lines and the control normal glial cells at different concentrations of ginkgolide B, and from this, the semi-lethal dose of different ginkgolide B on the cell lines was calculated; FIG. 2 shows the growth curve (U87) of glioma cells U87 with varying concentrations of ginkgolide B over time, showing that treatment with ginkgolide B at a semilethal concentration completely inhibited the growth of glioma cells, whereas treatment with ginkgolide B at a semilethal concentration 1/2 also partially inhibited the growth of glioma cells; FIG. 3 is a cell morphology observation, and the growth inhibition and the cell morphology change of the three glioma cell lines are realized by the ginkgolide B with different concentrations, so that the number of the cells is reduced and the cell morphology is shrunk and rounded or even floated under the action of the ginkgolide B; FIG. 4 shows cell migration, which is significantly inhibited by increasing ginkgolide B concentration. Under the treatment of high-concentration ginkgolide B, the healing of cell scratches becomes slow; FIG. 5 shows the effect of BrdU labeling on glioma cell proliferation of ginkgolide B, and the effect of Ki-67 labeling on non-resting state Brdu labeling on proliferative cells, which shows that both non-resting state and proliferative glioma cells are inhibited under treatment of ginkgolide B, and the corresponding quantitative results are shown in the bar chart of FIG. 5 (B). Meanwhile, as shown in fig. 6, it can be seen through animal experimental studies that the bilobalide B inhibits the growth of glioma cells not only effectively in vitro but also well in vivo formed glioma, and the corresponding quantitative results are shown in the histogram of fig. 6 (B).

Therefore, the ginkgolide B serving as an active substance has a good treatment effect on malignant glioma, is a natural product, can inhibit the migration and proliferation of glioma cells to different degrees, strictly controls the proliferation rate of the glioma cells, and has small side effect on organisms.

From the data associated with the above examples, it can be seen that the above requirements can be satisfied in all aspects only by the embodiments within the scope of the claims of the present invention, and that an optimized embodiment can be obtained, and that an inhibitor of the activity of a glioma cell containing bilobalide B with the optimum activity can be obtained. The change of the mixture ratio, the replacement/addition/subtraction of raw materials or the change of the feeding sequence can bring corresponding negative effects.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. Application of bilobalide B and its derivatives in preparing glioma cell activity inhibitor is provided.

2. The glioma cell activity inhibitor is characterized by comprising ginkgolide B and derivatives thereof, wherein the specific structural formula of the ginkgolide B is as follows:

3. the inhibitor of glioma cell activity of claim 2, wherein said glioma cells comprise one or more of glioma cell U-87MG, glioma cell U-251MG, or glioma cell a 172.

4. The use of the inhibitor of glioma cell activity of claim 2 or 3 in a chemotherapeutic drug for malignant brain glioma.

5. The use of claim 4, wherein said chemotherapeutic agent comprises one or more of ginkgolide B and pharmaceutically acceptable esters of said ginkgolide B.

6. The use according to claim 4, wherein the ginkgolide B is administered to the glioma cell A172 at a concentration of 1095-.

7. The use of claim 4, wherein the chemotherapeutic agent comprises an inhibitor of ginkgolide B and a pharmaceutically acceptable carrier and/or excipient.

8. The use of claim 7, wherein the chemotherapeutic agent is an oral or injectable formulation.

9. The pharmaceutical of claim 8, wherein the oral formulation is in the form of a capsule or tablet.

10. The medicament of claim 9, wherein the dosage of said oral formulation is about 5 to 5.2mg/kg body weight.