CN115737623A

CN115737623A - Application of mangiferin in preparation of medicine for treating osteosarcoma

Info

Publication number: CN115737623A
Application number: CN202210794318.6A
Authority: CN
Inventors: 王振新; 简明红; 张婳
Original assignee: Changchun Institute of Applied Chemistry of CAS
Current assignee: Changchun Institute of Applied Chemistry of CAS
Priority date: 2022-07-07
Filing date: 2022-07-07
Publication date: 2023-03-07

Abstract

The invention relates to the field of biological medicines, in particular to application of mangiferin in preparing a medicine for treating osteosarcoma. The invention screens matrix metalloproteinase inhibitor from compound in large scale by polypeptide microarray chip high throughput screening platform, wherein mangiferin is specificity inhibitor of matrix metalloproteinase-9, and compared with commercial matrix metalloproteinase inhibitor, the inhibition effect is obviously enhanced. The mangiferin has low cytotoxicity, can obviously inhibit the invasion of osteosarcoma cells U-2OS, and simultaneously effectively inhibit the growth and the remote metastasis of osteosarcoma, and the tumor inhibition rate of the mangiferin on the osteosarcoma can reach 35%. In long-term treatment, untreated mice die in 40 days, and mangiferin-treated osteosarcoma mice die in 48 days, which proves that the survival time of the mangiferin-treated mice is obviously prolonged, and the mangiferin can be used as a very effective medicament for treating osteosarcoma and has a good clinical application prospect.

Description

Application of mangiferin in preparation of medicine for treating osteosarcoma

Technical Field

The invention relates to the field of biological medicines, in particular to application of mangiferin in preparing a medicine for treating osteosarcoma.

Background

Mangiferin (mangiferin), also called skimmianin or mangiferin, is a carbon ketoside of tetrahydroxy pyridone, belongs to a flavonoid compound of the bispyridone, is mainly derived from dried rhizomes of perennial herb anemarrhena asphodeloides bunge of Liliaceae, leaves, fruits and barks of plants including almond and mango, roots of pterygophyllaceae and the like, has various pharmacological activities of relieving cough and asthma and eliminating phlegm, resisting inflammation and pain, protecting liver and benefiting gallbladder, resisting lipid peroxidation, resisting diabetes, resisting bacteria and viruses and the like, but has never found application in the aspect of resisting osteosarcoma.

Matrix metalloproteinases have increased activity in the growth, invasion and metastasis of tumors and in adverse prognostic processes. Osteosarcoma is a highly aggressive malignancy with high frequency of lung metastasis, poor prognosis and very low 5-year survival rate in patients. Therefore, matrix metalloprotease is considered as an important target for osteosarcoma diagnosis and anticancer drug development.

Osteosarcoma, a primary malignant bone tumor, occurs more frequently in adolescent and children populations, and is the most common bone tumor. It is a tumor bone-like tissue or bone tissue that develops from a mutation in the mesenchymal cell line, and is formed directly or indirectly through the cartilage stage. The prominent symptom of osteosarcoma is pain at the tumor site, which is caused by infiltration of tumor tissue and dissolution of cortical bone. When clearly diagnosed, the general condition is generally poor, manifested as fever, malaise, weight loss, anemia and even failure. Osteosarcoma may cause pain-avoiding claudication, limited joint movement and muscle atrophy. The conventional treatment technology is usually radical operation, namely amputation. Before amputation, stability chemotherapy or radiotherapy is generally carried out to reduce the tumor volume, reduce the uncertainty of the operation and improve the success rate of the operation; after amputation, consolidated chemotherapy or radiotherapy is performed to control tumor metastasis and improve patient survival.

Osteosarcoma is still the disease with the highest mortality rate of malignant tumors in children and adolescents at present, and the closer the tumor site is to the trunk, the higher the mortality rate. The relationship between the type of tumor and the degree of vascular abundance and prognosis is difficult to judge. Therefore, osteosarcoma remains a problem in current world oncology. In summary, to date, there is no ideal cure means and medical treatment technique in the clinical medical field of tumor for osteosarcoma, and mangiferin has never been used for treating osteosarcoma.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide an application of mangiferin in the preparation of a medicament for treating osteosarcoma.

The invention provides application of mangiferin in preparing a medicament for treating osteosarcoma.

In the use of the invention, the treatment comprises inhibiting matrix metalloproteinase activity of osteosarcoma cells.

In the use of the invention, the matrix metalloproteinase comprises matrix metalloproteinase-9. The mangiferin is a specific inhibitor of matrix metalloproteinase-9, and the activity of the matrix metalloproteinase is represented by the change value of fluorescence signals before and after the enzyme digestion reaction of a corresponding polypeptide substrate. The inhibitory properties of the compounds are indicated by the change in matrix metalloproteinase activity before and after addition of the compound. IC (integrated circuit) ₅₀ Refers to the concentration of a drug that has a 50% inhibitory effect on matrix metalloproteinase activity. The concentration (IC) of the semi-inhibitor of mangiferin of the present invention ₅₀ ) Are all 250nM.

In the use of the invention, the treatment comprises inhibiting osteosarcoma cell activity. The treatment effect of the medicine comprises that the tumor inhibition rate of mangiferin on osteosarcoma reaches 35%. The mangiferin can reduce the relative activity of matrix metalloproteinase-9, when the concentration of the mangiferin is 0nM, the relative activity of the matrix metalloproteinase-9 is close to 100%, and along with the increase of the concentration of the mangiferin, the relative activity of the matrix metalloproteinase-9 is gradually reduced to 0.

In the use of the invention, the treatment comprises inhibition of osteosarcoma cell invasion and/or inhibition of osteosarcoma metastasis. The inhibition of cell invasion or cell transfer is represented by inhibiting cell migration, the mobility of the cells of the mangiferin-treated mice is reduced to 75-80%, and the mobility of the cells of the control group which are not treated with mangiferin is 100%.

In the use of the invention, the treatment is inhibition of osteosarcoma growth, which includes inhibition of growth of osteosarcoma volume and/or inhibition of increase in osteosarcoma weight. Within 14 days, the relative tumor volume of the tumor of the control group mouse increases more quickly, the relative tumor volume reaches 40-45 in the fourteenth day, the tumor mass reaches 1.75-2 g, the relative tumor volume of the tumor of the mangiferin treatment group mouse increases slowly, the relative tumor volume is only 25-30 g in the fourteenth day, the tumor mass is only 1.0-1.25 g, and the tumor mass of the mangiferin treatment group is smaller.

In the use according to the invention, the treatment comprises prolonging the survival time and/or increasing the survival rate. The survival time of the control group mouse is only 40 days, while the survival time of the mangiferin treatment group mouse is 45-50 days, which prolongs the survival time of the mouse by 5-10 days.

In the application of the invention, the osteosarcoma is a tibia intermediate differentiated sarcoma.

In the application of the invention, the effective treatment concentration of mangiferin is 1.5mg/kg.

The invention also provides a medicine for treating osteosarcoma, which comprises mangiferin. Because in the U-2OS tumor-bearing mouse model of the invention, mangiferin has satisfactory anti-tumor effect and negligible toxicity. This indicates that mangiferin, as a matrix metalloproteinase inhibitor, is a potential drug for treating osteosarcoma.

The cells used in the examples of the present invention include: the breast cancer cells MDA-MB-231 and MCF-7, the kidney epithelial cell 293T, the colon epithelial cell NCM460, the liver cell HL-7702, the fibrosarcoma cell HT-1080, the colon cancer cell HCT-116 and the osteosarcoma cell U-2OS are all human cells, and the cell survival rate of the eight cells is over 90 percent after the eight cells are cultured with 2.5 mu M mangiferin for 24 hours, which indicates that the mangiferin has little toxicity to the cells.

The mangiferin has low cytotoxicity, can obviously inhibit the invasion of osteosarcoma cells U-2OS, effectively inhibit the growth and remote metastasis of osteosarcoma, and has the tumor inhibition rate of the mangiferin on the osteosarcoma of 35%.

The invention also provides a method for treating osteosarcoma, which comprises the step of administering the medicament.

The dosage form of the drug of the invention is oral preparation or injection, the administration mode of the drug of the invention can be gastrointestinal administration or parenteral administration, the parenteral administration is selected from sublingual administration preparation, intramuscular injection administration preparation, intravenous administration preparation, rectal administration preparation, mucosal administration preparation and respiratory tract administration preparation, and in some embodiments, intramuscular injection and mucosal administration are selected. In some embodiments, the injection method of the injection is once every two days for three consecutive days.

The solvent of the pharmaceutical preparation is selected from normal saline, PBS solution with pH value of 7.2-pH value of 7.4, PBS solution containing 0.05-5% of chitosan or sodium alginate, or glucose solution with concentration of 5%. Preferably, the pharmaceutical dosage form is a liquid injection preparation, and the solvent is physiological saline.

The invention provides a polypeptide microarray chip high-throughput screening compound based on a zinc oxide nanorod substrate modified by a poly (glycidyl methacrylate-co-hydroxyethyl methacrylate) brush for inhibiting the activity of matrix metalloproteinase, so as to realize osteosarcoma treatment, and the screening specifically comprises the following steps:

step 1: preparing a zinc oxide nanorod substrate chip substrate modified by a poly (glycidyl methacrylate-co-hydroxyethyl methacrylate) brush;

and 2, step: fixing a biotin-modified polypeptide substrate on the substrate in the step 1 to form a polypeptide microarray chip;

and step 3: culturing U-2OS osteosarcoma cells to obtain a cell culture medium;

and 4, step 4: reacting the U-2OS osteosarcoma cell culture medium obtained in the step (3) with different compounds and the polypeptide microarray chip prepared in the step (2) for 4 hours, and reacting with Cy3 modified avidin to obtain a fluorescence-labeled polypeptide microarray chip;

and 5: a decrease in the change in fluorescence is detected in step 4, indicating that the compound to be tested has activity in inhibiting matrix metalloproteases.

The compounds to be tested include: psoralen A, neodrynarium isoflavone, mangiferin, psoralen B, hesperetin, baicalein, wogonin, scutellarin, baicalin, silybin, tectoridin, tectorigenin, formononetin, biochanin A, formononetin, isosilybin, silydianin, silychristin, sinensetin, vitexin, apigenin, naringin chalcone, licochalcone, liquiritin, rutin, liquiritin, isoliquiritin, nobiletin, isoliquiritigenin, hesperidin, naringin, mullein, daidzein, daidzin, genistein, sophoricoside, glycitin, glycitein, ginkgetin, naringenin, isorhamnetin, morin, kaempferol, douglas fir, dihydromyricetin, quercetin, myricitrin and neodecirin.

The polypeptide substrate comprises S0, S1, S2, S3, S7, S9, S13; the S0 has an amino acid sequence shown as SEQ ID No.1 (CGGKGDEDEDEDEDEDEDEDEGK), the S1 has an amino acid sequence shown as SEQ ID No.2 (CGGKGVPMSMRGK), the S2 has an amino acid sequence shown as SEQ ID No.3 (CGGKGAEGFFSARK), the S3 has an amino acid sequence shown as SEQ ID No.4 (CGGKGRPKPVE-Nva-WRGK), the S7 has an amino acid sequence shown as SEQ ID No.5 (CGGKGRPLALWRSGK), the S9 has an amino acid sequence shown as SEQ ID No.6 (CGGKGPRSLSGGK), and the S13 has an amino acid sequence shown as SEQ ID No.7 (CGGKGGMRGK).

According to the invention, through an MTT (methyl thiazolyl tetrazolium) experiment and a scratch experiment, the influence of mangiferin on the cytotoxicity and invasiveness of U-2OS is found; in addition, the invention also evaluates the tumor inhibition effect of mangiferin through an osteosarcoma U-2OS tumor-bearing mouse model. The tumor growth in the present invention is expressed in terms of the relative tumor volume, i.e., the ratio of the volume of the drug treated to the tumor volume before treatment.

The invention discovers that mangiferin is a specific inhibitor of matrix metalloproteinase-9, and the concentration (IC) of a semi-inhibitor ₅₀ ) Is 250nM, the inhibition is significantly enhanced compared to commercial matrix metalloproteinase inhibitors. The mangiferin has low cytotoxicity, can obviously inhibit the invasion of osteosarcoma cells U-2OS, and simultaneously effectively inhibit the growth and remote metastasis of osteosarcoma, and the tumor inhibition rate of the mangiferin on the osteosarcoma can reach 35%. In the long-term treatment of osteosarcoma, untreated mice die within 40 days, and mangiferin-treated osteosarcoma mice die within 48 days, which proves that the survival time of the mangiferin-treated mice is obviously prolonged, and the result can prove that mangiferin can be used as a very effective medicament for treating osteosarcoma and has a good clinical application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts according to the drawings:

FIG. 1 is a schematic diagram showing a screening scheme for a matrix metalloproteinase inhibitor of the present invention;

FIG. 2 shows the inhibitory properties of 51 compounds of the present invention on the activity of six matrix metalloproteinases described above;

FIG. 3 shows the effect of mangiferin concentration on matrix metalloproteinase-9 activity in the present invention;

FIG. 4 shows the effect of mangiferin on cell activity;

FIG. 5 shows the effect of mangiferin on the invasiveness of U-2OS in osteosarcoma cells; a in FIG. 5 shows the effect of the control group and mangiferin treatment group on the U-2OS cell invasion ability of osteosarcoma cells in the scratch test; b in fig. 5 illustrates the effect of the specific quantification control group and mangiferin treatment group on cell mobility;

FIG. 6 shows the tumor suppression effect of mangiferin on osteosarcoma U-2OS mice; a in fig. 6 indicates the time at which the mouse injected the drug; b in fig. 6 shows the relative tumor volumes of the control group and mangiferin treated group over time within 20 days; c in fig. 6 shows the tumor mass of the control group and the mangiferin treated group;

FIG. 7 shows the long-term tumor suppression effect of mangiferin in osteosarcoma U-2OS mice in the present invention.

Detailed Description

The invention provides application of mangiferin in preparing a medicament for treating osteosarcoma, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the application. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Table 1: polypeptide sequences used in the study

Polypeptide name	Sequence of ^a
		S0(SEQ ID NO.1)	CGGKGDEDEDEGK(biotin)
S1(SEQ ID NO.2)	CGGKGVPMSMRGK(biotin)
		S2(SEQ ID NO.3)	CGGKGAEGFFSARK(biotin)
S3(SEQ ID NO.4)	CGGKGRPKPVE-Nva-WRGK(biotin)
		S7(SEQ ID NO.5)	CGGKGRPLALWRSGK(biotin)
S9(SEQ ID NO.6)	CGGKGPRSLSGK(biotin)
		S13(SEQ ID NO.7)	CGGKGPLGMRGK(biotin)

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

Experimental methods

1. Preparation of Poly (glycidyl methacrylate-co-hydroxyethyl methacrylate) Brush-modified Zinc oxide nanorod substrate

1) Soaking the optical-grade glass sheet in 50mM potassium permanganate aqueous solution containing n-butanol with the volume fraction of 0.25% for 1h, and modifying the surface with alkaline manganese oxide (MnOOH);

2) Soaking the substrate modified by alkaline manganese oxide in a zinc sulfate solution containing 10% of ethanolamine, 4% of ammonium hydroxide and 0.1M in volume fraction, and growing a zinc oxide nanorod on the surface of the substrate to prepare the substrate modified by the zinc oxide nanorod;

3) Soaking the zinc oxide nanorod substrate in an absolute ethyl alcohol solution containing 5% (3-aminopropyl) triethoxysilane by volume fraction for reaction for 1h to obtain an amino-modified zinc oxide nanorod substrate;

4) And (2) putting the amino modified zinc oxide nanorod substrate into an anhydrous dichloromethane solution containing 5% of alpha-bromoisobutyryl bromide and 5% of triethylamine by volume fraction, reacting at 0 ℃ for 30min, and then reacting at 25 ℃ for 2h to obtain the zinc oxide nanorod substrate with the fixed initiator.

5) Putting the zinc oxide nanorod substrate modified by the initiator into a water/methanol (volume fraction is 50%) solution containing 1% of glycidyl methacrylate, 10% of hydroxyethyl methacrylate, 5mg/mL of cuprous bromide and 10.4mg/mL of 2,2' -bipyridyl, reacting for 9 hours at 30 ℃, sequentially washing with methanol and water, and then putting into a refrigerator at 4 ℃ for storage. And obtaining the zinc oxide nanorod substrate modified by the poly (glycidyl methacrylate-co-hydroxyethyl methacrylate) brush.

2. Production of polypeptide microarray chips

Preparing a polypeptide microarray chip by using a zinc oxide nanorod substrate modified by a poly (glycidyl methacrylate-co-hydroxyethyl methacrylate) brush and a SmartArrayer 136 biochip spotting system:

1) Sample application: the sample amount is 1 nL/point; in order to obtain a good and uniform array point and keep the activity of the biological molecules, the selected sample solution comprises the following components: 0.3M phosphate buffer solution (pH = 8.5) containing 20 μ g/mL bovine serum albumin, 20% PEG-2000,0.15m sodium chloride by mass fraction; and (3) using the spotting liquid to perform spotting on the zinc oxide nanorod substrate modified by the poly (glycidyl methacrylate-co-hydroxyethyl methacrylate) brush, and performing vacuum drying reaction for 12h at 30 ℃ after spotting to complete the fixation of the polypeptide on the zinc oxide nanorod substrate modified by the poly (glycidyl methacrylate-co-hydroxyethyl methacrylate) brush. Seven biotin-modified polypeptides, wherein one control peptide and six control peptides respectively have the amino acid sequences of matrix metalloproteinase-1, matrix metalloproteinase-2, matrix metalloproteinase-3, matrix metalloproteinase-7, matrix metalloproteinase-9 and matrix metalloproteinase-13 specific recognition sequences as shown in a sequence table 1.

2) Cleaning and sealing the polypeptide microarray chip: after the above-mentioned polypeptide microarray chip was dried in vacuum for 12 hours, the non-immobilized polypeptide substrate was washed with 0.05M phosphate buffer (pH = 7.5) containing 1% bovine serum albumin, and then unreacted epoxy groups were blocked with 0.05M phosphate buffer (pH = 7.5) containing 1% bovine serum albumin, 1% ethanolamine, and 0.15M sodium chloride to obtain a polypeptide microarray chip. The washing with a buffer solution containing 0.1% by volume of polyoxyethylene octylphenyl ether, 0.15M sodium chloride, 0.05M tris (hydroxymethyl) aminomethane (pH = 7.5), and a buffer solution containing 0.05% by volume of polyoxyethylene lauryl ether, 0.15M sodium chloride, 10mM calcium chloride, and 25mM tris (hydroxymethyl) aminomethane (pH = 7.5) was continued. Finally, the polypeptide microarray chip which is sealed and cleaned is divided into 12 arrays by a polytetrafluoroethylene fence.

3. Cell culture and matrix metalloproteinase harvesting

Osteosarcoma cell U-2OS was obtained from the cell bank of the Committee for type culture Collection of the Chinese academy of sciences at 37 deg.C, 5% ₂ Under the conditions of (1), the cells were cultured in McCoy's 5A medium containing 10% fetal bovine serum. After the cells were confluent, 5000U-2 OS cells per well were seeded in 48-well cell culture plates and cultured for 24h in McCoy's 5A medium containing 10% fetal bovine serum. Then washed with PBS and cultured for an additional 24 hours by adding fresh McCoy's 5A medium. The cell culture medium after culturing the U-2OS cells was centrifuged at 1000rpm at 4 ℃ for 10min to obtain an osteosarcoma U-2OS cell culture medium containing matrix metalloproteinase.

4. Screening of matrix metalloproteinase activity inhibitors from compounds

1) 30 mu L of osteosarcoma U-2OS cell culture medium supernatant and 51 compounds are respectively mixed and added into the polypeptide microarray chip which is sealed and cleaned in the step 4, and the reaction is carried out for 4 hours at 37 ℃ and 80% humidity. Then, the mixture was washed with 0.1% of polyoxyethylene octylphenyl ether in tris buffer (pH = 7.5), tris buffer, deionized water and centrifuged.

51 compounds comprising: psoralen A, neodrynarium isoflavone, mangiferin, psoralen B, hesperetin, baicalein, wogonin, scutellarin, baicalin, silybin, tectoridin, tectorigenin, formononetin, biochanin A, formononetin, isosilybin, silydianin, silychristin, sinensetin, vitexin, apigenin, naringin chalcone, licochalcone, liquiritin, rutin, liquiritin, isoliquiritin, nobiletin, isoliquiritigenin, hesperidin, naringin, mullein, daidzein, daidzin, genistein, sophoricoside, glycitin, glycitein, ginkgetin, naringenin, isorhamnetin, morin, kaempferol, douglas fir, dihydromyricetin, quercetin, myricitrin and neodecirin.

2) After the polypeptide microarray chip is labeled by the avidin modified by 50nM Cy3 after the reaction, a phosphate buffer solution, deionized water and centrifugal drying are sequentially used for cleaning and centrifugal drying to obtain the polypeptide microarray chip labeled by fluorescence, and then a phosphate buffer solution containing 0.1 percent of Tween-20, a phosphate buffer solution and deionized water are sequentially used for cleaning and centrifugal drying. The fluorescence labeled polypeptide microarray chip is put into a microarray scanner (such as LuxScan-10K/A type microarray scanner produced by Beijing Boao biotechnology, inc.) to obtain the fluorescence detection signal of the polypeptide microarray chip.

The matrix metalloproteinase inhibitor screening of the invention is shown in figure 1.

The activity of matrix metalloprotease is represented by the change of fluorescence signal before and after the corresponding polypeptide substrate enzyme cleavage reaction.

The inhibitory properties of the compounds are indicated by the change in matrix metalloproteinase activity before and after addition of the compound.

IC ₅₀ Refers to the concentration of a drug that has a 50% inhibitory effect on matrix metalloproteinase activity.

According to the above experimental procedures, the inhibitory effect of the compound obtained in the present invention on the activity of matrix metalloproteinase is shown in FIG. 2. FIG. 2 is a chart showing the inhibitory effect of 51 compounds used in the present invention on matrix metalloproteinase-1, matrix metalloproteinase-2, matrix metalloproteinase-3, matrix metalloproteinase-7, matrix metalloproteinase-9 and matrix metalloproteinase-13 secreted from osteosarcoma U-2OS cells. Wherein, the mangiferin has stronger specific inhibition effect on the activity of matrix metalloproteinase-9, and the inhibitor curve of the mangiferin and the matrix metalloproteinase in the invention is shown in figure 3. Fig. 3 is an inhibitor curve of the activity of matrix metalloproteinase obtained by the invention along with the change of the concentration of mangiferin, wherein the abscissa of the graph is the concentration of mangiferin, the ordinate is the relative activity of matrix metalloproteinase, and the relative activity of matrix metalloproteinase is reduced along with the increase of the concentration of the compound. Wherein mangiferin is specific inhibitor of matrix metalloproteinase-9, and semi-Inhibitor Concentration (IC) ₅₀ ) 250nM each, and the mangiferin concentrations added to the experiments were 1nM,5nM,10nM,50nM,100nM,500nM and 1000nM.

5. Cell viability assay

1) Breast cancer cells MDA-MB-231 and MCF-7, kidney epithelial cell 293T, colon epithelial cell NCM460, liver cell HL-7702, fibrosarcoma cell HT-1080, colon cancer cell HCT-116, and osteosarcoma cell U-2OS were all human cells and purchased from cell banks of the China academy of sciences type culture Collection. At 37 ℃ 5% CO ₂ In a medium containing 10% fetal bovine serum, wherein MDA-MB-231, MCF-7, 293T, NCM460, HL-7702 are cultured in a DMEM medium, HT-1080 is cultured in an RPIM 1640 medium, and HCT-116 and U-2OS are cultured in a McCoy's 5A medium.

2) The mangiferin screened by the method 4 is used for detecting cytotoxicity by using the traditional MTT assay. The above eight cells were inoculated into 96-well cell culture plates at 10000 cells per well, and the cell density reached 80%. The test drugs were added to the cells after preparing solutions of 0.1. Mu.M, 0.25. Mu.M, 0.5. Mu.M, 1. Mu.M, and 2.5. Mu.M, respectively, using the culture medium of the corresponding cells, and cultured for 24 hours. After washing with PBS solution, 100. Mu.L of 5mg/mL MTT solution was added thereto and reacted for 4 hours. Next, the supernatant was removed, 100. Mu.L of dimethyl sulfoxide was added, shaken for 10min, and the light intensity (OD) value was measured using a Powerwave XS2 plate reader.

The cell viability is the ratio of the OD value of the cells cultured by adding the compound and the OD value of the cells cultured without adding mangiferin.

The results obtained according to the invention according to the above experimental procedure are shown in FIG. 4. FIG. 4 shows the effect of mangiferin concentration on the viability of U-2OS cells of osteosarcoma cells obtained by the present invention. In the invention, after the eight cells are cultured with 2.5 mu M mangiferin for 24 hours, the cell survival rate exceeds 90 percent, which shows that the mangiferin has almost no toxicity to osteosarcoma U-2OS cells in the invention.

6. Effect of Mangiferin on cell invasiveness

The osteosarcoma U-2OS cells were seeded in a 6-well plate at 80000 cells per well, and when the cells grew to fuse to a monolayer state, a scratch was artificially made on the fused monolayer cells with a 200. Mu.L tip of a pipette tip, the suspension cells were washed off with PBS, and McCoy's 5A cell culture medium containing 2.5. Mu.M mangiferin was added for co-culture for 24h. After washing with PBS, 1mL of Hoechst 33342 (4. Mu.g/mL) was added for staining, and imaging was performed with a Nikon Ti-S fluorescence microscope.

Cell migration was calculated as the percentage of the area of the scratch reduced.

The results obtained according to the invention are shown in FIG. 5, following the above experimental procedures. FIG. 5 shows the effect of mangiferin obtained by the present invention on the invasion ability of osteosarcoma cell U-2OS cell. In the present invention, as shown in fig. 5 (a) by bright field and fluorescence imaging, scratches between cells were almost closed after culturing with the medium alone for 24 hours. In addition, after 2.5 mu M of mangiferin is added into the culture medium respectively and is cultured with the cells for 24 hours, scratches among the cells are still clearly visible. The calculation of the scratch change area in the present invention can obtain the result shown in fig. 5 (b).

7. In vivo anti-osteosarcoma study

Female NOD/SCID mice and BALB/c nude mice were injected subcutaneously with 100. Mu.L of a solution containing 3.0X 10 ⁶ A mouse model of osteosarcoma U-2OS was constructed from PBS solution of individual U-2OS cells. When tumors grew to 3-5 mm, NOD/SCID mice and BALB/c nude mice were divided into 2 groups, and 100. Mu.L of PBS solution and 100. Mu.L of mangiferin (1.5 mg/kg) in PBS solution were injected through the tail vein once every two days for three consecutive times. Osteosarcoma models constructed from NOD/SCID mice were sacrificed throughout 14 days after drug injection was completed. The osteosarcoma model constructed in BALB/c nude mice was monitored for the time to death after drug treatment.

The tumor growth in the present invention is expressed in terms of the relative tumor volume, i.e., the ratio of the volume of the drug treated to the tumor volume before treatment.

According to the above experimental procedures, the result of mangiferin inhibiting osteosarcoma obtained by the present invention is shown in fig. 6. FIG. 6 (b) is the tumor growth curves of NOD/SCID osteosarcoma mice injected with PBS and mangiferin-containing PBS2 groups within 14 days of the initial tail vein injection and drug treatment, wherein the inset is the bright field image of the tumor of the corresponding 2 groups of NOD/SCID mice at the time of sacrifice. The tumor inhibition rate of mangiferin on U-2OS osteosarcoma mice is 35 percent

According to the experimental steps, the treatment effect of mangiferin on long-term osteosarcoma is shown in fig. 7. FIG. 7 shows survival curves of 2 groups of mice within 70 days of initial tail vein injection and drug treatment of BALB/c osteosarcoma mice. The osteosarcoma mice without drug treatment in the invention all die within 40 days, and the mangiferin treated mice all survive within 41 days after treatment.

In the U-2OS tumor-bearing mouse model, mangiferin has a satisfactory anti-tumor effect and negligible toxicity. This indicates that mangiferin, as a matrix metalloproteinase inhibitor, is a potential drug for treating osteosarcoma.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of mangiferin in preparing medicine for treating osteosarcoma is provided.

2. The use of claim 1, wherein the treatment comprises inhibiting matrix metalloproteinase activity of osteosarcoma cells.

3. Use according to claim 2, wherein the matrix metalloproteinase comprises matrix metalloproteinase-9.

4. The use of claim 1 or 2, wherein the treatment comprises inhibiting osteosarcoma cell activity.

5. The use of claim 1, 2 or 4, wherein the treatment comprises inhibiting osteosarcoma cell invasion and/or inhibiting osteosarcoma metastasis.

6. The use of claim 1, 2, 4 or 5, wherein the treatment comprises inhibiting the growth of osteosarcoma.

7. The use according to any one of claims 1, 2 or 4 to 6, wherein the treatment comprises prolonging survival and/or increasing survival.

8. The use of claim 1, 2, 3, 4 or 6, wherein the osteosarcoma is a moderately differentiated sarcoma of the tibia.

9. The use of claim 1, wherein the therapeutically effective concentration of mangiferin is 1.5mg/kg.

10. A medicine for treating osteosarcoma is characterized by comprising mangiferin.