CN107050008B - Application of gray folic acid in preparation of medicine for treating osteosarcoma - Google Patents

Abstract

The application of the folic acid disclosed in the invention in preparing a medicine for treating osteosarcoma, the concentration of the folic acid in the medicine is not less than 50 μmol/L; the administration mode of the medicine is intratumoral injection of the osteosarcoma; the injection of the folic acid in the osteosarcoma per cubic millimeter The dose is 0.1ml. The invention proposes new uses of folic acid. These uses are based on the discovery and confirmation of new functions. The intratumoral injection of folic acid can effectively kill osteosarcoma cells, significantly inhibit the growth of osteosarcoma, and at the same time, there is no obvious side effect. Osteosarcoma's high killing effect and low toxicity to the body make it a good drug for the treatment of osteosarcoma and other tumors.

Description

The application of gray folic acid in the preparation of drugs for the treatment of osteosarcoma

technical field

The invention belongs to the technical field of application of folic acid, in particular to the application of folic acid in preparing a medicine for treating osteosarcoma.

Background technique

Osteosarcoma mostly occurs in young people and children, with a high degree of malignancy and a poor prognosis. Despite continuous improvements in clinical treatment, osteosarcoma survival has not improved further over the past three decades. Chemotherapy is the most important treatment method for osteosarcoma besides surgery. Although increasing the dose of chemotherapy drugs can achieve a more significant effect of killing tumor cells, the currently used chemotherapy drugs have unbearable side effects when used in high doses. , which limits its application in killing tumor cells. Therefore, it is of great significance to find safer and more effective antitumor drugs.

Grifolic acid is a natural product extracted from Glycystis chinensis. There are few reports on the pharmacological effects of grifolic acid. It is generally believed that grifolic acid is an agonist of free fatty acid receptors, which can stimulate G protein. Conjugated receptor 120 (GPR120). Grifolin, an analog of grifolic acid, has been found to have significant antitumor effects and can kill a variety of tumor cells, but the role of grifolic acid is unclear.

Studies have shown that gray folic acid can inhibit the production of ATP in cells. The level of intracellular ATP is very important for cell activity. Intracellular ATP is produced by mitochondria, and mitochondria convert acetyl-CoA derived from glucose and fatty acids through oxidative phosphorylation. Through the tricarboxylic acid cycle, CO ₂ is generated, and hydrogen ions are transmitted along the respiratory chain through the hydrogen transfer body. During this process, the hydrogen ion concentration difference between the two sides of the mitochondrial inner membrane is established, and this potential energy difference is used for ATP generation. The final oxidation produces water. Mitochondrial membrane potential is the energy source to maintain ATP production, and the loss of membrane potential will lead to reduced ATP production, which in turn leads to cell death due to energy shortage. Although gray folic acid can kill tumor cells by inhibiting the production of cellular ATP, it is still unknown whether gray folic acid has the effect of killing tumors in vivo and whether it has obvious toxic side effects.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an application of griffinic acid in the preparation of a medicament for treating osteosarcoma, which provides a new idea for inhibiting the proliferation of osteosarcoma cells and effectively killing osteosarcoma cells.

The technical solution adopted in the present invention is the application of griffinic acid in the preparation of a medicament for treating osteosarcoma.

Another technical solution adopted in the present invention is to prepare a medicament for treating osteosarcoma, and the medicament contains a gray folic acid component.

The present invention is also characterized in that,

The concentration of gray folic acid in the drug is not less than 50 μmol/L.

The drug is administered by intratumoral injection in osteosarcoma.

The injected dose of folic acid is: 0.1 ml of folic acid per cubic millimeter of osteosarcoma.

The beneficial effects of the present invention are as follows: the present invention proposes new uses of gray folic acid, which are discovered and confirmed based on its new functions. Intratumoral injection of folic acid can effectively kill osteosarcoma cells, significantly inhibit the growth of osteosarcoma, and have no obvious side effects. good medicine.

Description of drawings

Figure 1: Statistical results of the in vitro killing of various osteosarcoma cells by folic acid: After different osteosarcoma cells were treated with different concentrations of folic acid for 2 hours, 6 hours, 12 hours and 24 hours, the viability of osteosarcoma cells relative to the injection of folic acid Change ratio of precellular activity; A: Human MG-63 cells; B: Human U-2OS cells; C: Human Saos-2 cells; D: Mouse K7M2 cells; the number of cells in each group is n=12;

Figure 2: Statistical results of the killing effect of glycine folic acid injected at different sites on osteosarcoma cells in vivo: the two groups of mice were injected with glycine folic acid at different sites respectively, and after 7 days of injection: the comparison of tumor volume with the blank control group of mice (Picture A); the results of the proportion of tumor cells in the tumor obtained by analyzing the tumor tissue by tissue section staining (Picture B); the number of mice in each group above is 4;

Figure 3: Toxic and side effects of intratumoral injection of folic acid on the tissue morphology of different organs in mice: the difference between the control group and the experimental group was shown by hematoxylin-eosin (HE) staining analysis of tissue sections. The control group was not injected with folic acid. HE staining results of mouse organs in the experimental group, the experimental group is the HE staining results of the mouse organs injected with gray folic acid; A: the liver of the control group; B: the liver of the experimental group; C: the kidney of the control group; D: Kidneys of mice in experimental group; E: lungs of mice in control group; F: lungs of mice in experimental group; G: heart of mice in control group; H: heart of mice in experimental group;

Figure 4: Statistical results of intratumoral injection of glycine folic acid on the blood indexes of mice: the comparison of blood indexes in the control group with intratumoral injection of glycine folic acid and no injection of glycine folic acid in mice, A: number of red blood cells; B: number of white blood cells ; C: platelet count; D: total plasma protein; E: alanine aminotransferase; F: aspartate aminotransferase;

Figure 5: The effect of folic acid on the survival time of osteosarcoma-burdened mice: the survival curves of three groups of mice were analyzed by log-rank statistics. The three groups of mice were: mice without injection of folic acid, intravenous For the mice injected with folic acid and the mice injected with folic acid intratumorally, the number of mice in each of the above three groups was 16.

Detailed ways

The present invention will be further described in detail below with reference to specific examples, and the application of glycine folic acid in the preparation of a medicament for the treatment of osteosarcoma.

1. Cell culture: A variety of osteosarcoma cells derived from human and mouse were used to detect the effect of gray folic acid. Human MG-63 cells, human U-2OS cells, human Saos-2 cells and mouse K7M2 cells used in the present invention are all used for detection. All the above cells were cultured in DMEM medium containing 10% fetal bovine serum and cultured in a 5% _CO2 incubator at 37°C, the medium was changed every 2-3 days, and the cells were passaged after growing to 80%. Passaging After digestion with 0.25% trypsin + 0.02% EDTA, the cells were passaged at a ratio of 1:3-5. Culture medium, serum and digestive enzymes are products of Gibco Company.

2. Detection of cell activity: The above-mentioned cells were planted in 96-well culture plates, and after they had grown to 90%, they were replaced with serum-free culture medium, and 0 μM, 1 μM, 3 μM, 10 μM, and 30 μM were added with different concentrations of ash. Folic acid was incubated for 2 hours, 6 hours, 12 hours and 24 hours; then thiazolyl blue MTT (final concentration 0.5mg/ml) was added, MTT can penetrate the cell membrane and enter the cell, and the succinyl dehydrogenase in the mitochondria of living cells can make the external The source MTT was reduced to blue-purple needle-like crystals that were insoluble in water and deposited in the cells; after MTT was incubated for 4 hours, the supernatant was completely removed, and 100 μl of DMSO was added to each well, and DMSO could dissolve the crystals. Then, the absorbance (OD value) was measured at 560 nm using an enzyme-linked immunosorbent assay, which also reflected the number of cells.

The OD value of each group of cells was statistically analyzed by variance analysis. The specific results are shown in Figure 1: the concentration of 30 μM folic acid can significantly reduce human MG-63 cells and human U-2OS cells after two hours of treatment. , the activity of human Saos-2 cells and mouse K7M2 cells, and basically all killed osteosarcoma cells after 12 hours of incubation; the concentration of 3 μM and 10 μM folic acid also had the effect of killing osteosarcoma cells, but the dose was different. There is a certain dependence; the concentration of gray folic acid as low as 1 μM has no significant difference compared with the 0 μM control group within 24 hours, and the killing effect on osteosarcoma cells is not obvious. It can be seen that the concentration of 3 μmol-30 μmol of gray folic acid on the dose-dependent and time-dependent decreased significantly, the activity of the detected cells also gradually decreased.

3. Preparation and grouping of osteosarcoma mouse models: K7M2 mouse osteosarcoma cells were collected and concentrated to 5×10 ⁷ cells/ml. Balb/c mice were anesthetized by isoflurane inhalation, localized for disinfection, and 100 microliters of K7M2 cell suspension was subcutaneously injected into the back. After 14 days of injection, the local bulge was checked, and the following experiments were performed on mice with tumors: the first group was the control group without treatment; the second group was the group of mice injected with gray folic acid through the tail vein after anesthesia, and the injection dose was 50μmol/L, 0.5ml was injected; the third group was injected with 50μmol/L of gray folic acid and 0.5ml by intratumoral injection of micro-injector after anesthesia.

After the treatment, the mice were placed in the animal room after being awake and recovered. 7 days after the injection of gray folic acid, 4 mice were taken out from each group. After anesthesia, blood was collected from the heart, followed by paraformaldehyde perfusion fixation, and the size of the tumor was measured, as shown in the figure. 2 As shown in Figure A, the volume of osteosarcoma in the mice in the intravenous injection of folic acid group did not change significantly compared with the control group, and the volume of osteosarcoma in the mice in the intratumoral injection of folic acid also did not significantly decrease; Figure B in Figure 2 As shown, the analysis of tumor tissue by tissue section staining showed that the osteosarcoma cells of the mice in the intravenous injection of glycine folic acid group accounted for 92% of the tumor area, which was not significantly different from 90% of the control group. The tumor tissue was mainly composed of necrotic tissue, and osteosarcoma cells accounted for 36% of the tumor area, which was significantly less than the control group.

The tumor bodies and other important organs of the above-mentioned mice, including liver, heart, kidney, and lung, were embedded and sliced, and analyzed by hematoxylin-eosin (HE) staining; Protein, aspartate aminotransferase, alanine aminotransferase and other blood index levels were measured; the rest of the mice continued to be raised, the survival of the mice was observed every day, and the number of dead mice was recorded.

4. Hematoxylin-eosin (HE) staining: After the above mouse tissue sections were dewaxed and rehydrated with xylene, they were stained with hematoxylin for 1 min, rinsed with tap water for 1 min, differentiated with 1% hydrochloric acid alcohol for 1 min, and washed with tap water for 1 min. , 1% dilute ammonia water against blue 0.5mim, washed with tap water for 1min, then stained with eosin for 1min, washed with tap water, dehydrated with alcohol, transparent with xylene, sealed with neutral gum, and photographed under an ordinary light microscope.

As shown in Figure 3, the hematoxylin-eosin (HE) staining analysis of tissue sections showed that the tissue structures of the liver, kidney, lung and heart of the mice in the intratumoral injection of gray folic acid group were intact, and there was no significant difference from the control group.

5. Blood cell count and blood biochemical index detection: Take 50 microliters of blood and add it to a hemocytometer to count red blood cells, white blood cells and platelets; the rest of the blood samples are centrifuged to collect plasma, and a total protein detection kit (biuret colorimetry) is used. Plasma total protein levels were determined; plasma aspartate aminotransferase and alanine aminotransferase levels were also detected using assay kits for aspartate aminotransferase and alanine aminotransferase.

As shown in Figure 4, the levels of red blood cells, white blood cells, platelets, plasma total protein, alanine aminotransferase, and aspartate aminotransferase of the mice in the intratumoral injection of gray folic acid group were not significantly different from those in the control group.

6. Survival monitoring of mice: continue to raise, observe the survival of mice every day, and draw the curve shown in Figure 5 by recording the number of mice that survived. There was no significant difference between the two groups; the number of deaths of mice in the intratumoral injection of folic acid was significantly lower than that of the control group. Log-rank statistical analysis was used to analyze the survival time of mice in the intratumoral injection of folic acid. Compared with the control group and tail vein injection of gray folic acid group were significantly prolonged.

Claims (1)

1. Application of gray folic acid in preparing medicine for treating osteosarcoma is provided.

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