CN107823211A - Application of the gucosamine in preparing ionising radiation and causing induced lung injury protective agents - Google Patents

Abstract

The present invention relates to the field of new uses of medicines, in particular to the application of glucosamine in the preparation of drugs for the prevention and treatment of radiation-induced lung injury caused by ionizing radiation. The structural formula of the glucosamine is as follows. The glucosamine described in the present invention is used as a drug for the prevention and treatment of radiation-induced lung injury caused by ionizing radiation: it has little toxic and side effects and significant curative effect. It can significantly protect the lung tissue and inhibit the interepithelial interepithelium of the lung tissue when administered at a dose of 100 mg/kg/d 3 days before irradiation. The qualitative transformation shows that glucosamine is unique in the protection of ionizing radiation-induced radiation-induced lung injury, and has broad application prospects in the field of medicine in my country.

Description

Application of glucosamine in the preparation of drugs for the prevention and treatment of radiation-induced lung injury caused by ionizing radiation

technical field

The invention relates to the field of new applications of medicines, in particular to the application of glucosamine in the preparation of medicines for the prevention and treatment of radiation-induced lung injury caused by ionizing radiation.

Background technique

Radiation lung injury frequently occurs after radiation therapy for thoracic tumors. Radiation therapy is one of the most important treatments for thoracic tumors, and radiation lung injury, including radiation pneumonitis and radiation pulmonary fibrosis, is a common serious complication and dose-limiting factor in radiotherapy for thoracic tumors. The main clinical manifestations of radiation-induced lung injury are inflammatory infiltration of the alveolar interstitium, progressive dyspnea, deterioration of lung function, and eventually respiratory failure. The occurrence of radiation lung injury greatly reduces the prognosis and survival rate of patients receiving chest irradiation. Therefore, the protection and treatment of radiation-induced lung injury caused by radiotherapy for thoracic tumors has extremely important medical significance. Currently, there is no effective treatment except for symptomatic treatment with glucocorticoids. There is no unified conclusion in the academic circles about the cause of radiation-induced lung injury. Most people think that it is due to the immune imbalance of various inflammatory cells, fibroblasts and related cytokines induced by ionizing radiation, resulting in the excessive proliferation and migration of fibroblasts and the deposition of extracellular matrix (ECM), thus causing damage to the body . Currently, there is no effective protective and therapeutic drug for radiation-induced lung injury. While traditional radioprotective drugs play a radioprotective role in normal lung tissue, they also provide a certain protective effect on tumor cells. The characteristics of this class of drugs greatly reduce the effect of tumor radiotherapy, which restricts its application in chest radiotherapy for clinical tumor patients. Therefore, in the clinical treatment of radiation-induced lung injury, it is urgent to find a new type of protective drug with significant curative effect, low toxicity and high safety. At present, glucocorticoids are mainly used clinically to alleviate the acute inflammatory phase of radiation-induced lung injury, but this drug has no effect on long-term radiation-induced pulmonary fibrosis. At the same time, its severe side effects also limit the scope and dosage of clinical use of this drug . In animal experiments, the radioprotective drug WR-2721 can effectively prevent and reduce the acute inflammatory response of radiation-induced lung injury. However, the drug has no obvious effect on long-term radiation pulmonary fibrosis and has relatively strong biological toxicity, which limits its clinical application. In addition, experimental research on drugs for the prevention and treatment of radiation-induced lung injury mainly focuses on antioxidants, gene therapy, and stem cell therapy. Therefore, finding a drug with high efficiency and low toxicity, which exerts a radioprotective effect on normal lung tissue and does not protect tumor cells or even has tumor killing effect, has always been an urgent problem to be solved in the research of radiation-induced lung injury.

Glucosamine (Glucosamine) exists widely in nature, usually in the form of N-acetyl derivatives (such as chitin) or in the form of N-sulfate and N-acetyl-3-O-lactate In polysaccharides and bound polysaccharides of animal origin. It is a compound in which one hydroxyl group of glucose is replaced by an amino group. Its molecular formula is C ₆ H ₁₃ O ₅ N, commonly known as amino sugar, or ammonia sugar for short. It is a substance synthesized in the human body, an important nutrient for the formation of chondrocytes, and a natural tissue component of healthy articular cartilage. As we grow older, the lack of glucosamine in the body becomes more and more serious, and the articular cartilage continues to degenerate and wear away. A large number of medical studies in the United States, Europe and Japan have shown that glucosamine can help repair and maintain cartilage, and can stimulate the growth of chondrocytes. It has been widely used in the daily health care of the human body. Glucosamine has anti-inflammatory and anti-oxidative effects, and its toxic and side effects are found to be very small, showing very good biological safety. Recent studies have found that glucosamine has a significant killing effect on a variety of human tumor cells. Combined with the characteristics of glucosamine, the drug has great potential application value in clinical radiation lung injury.

There is no report about glucosamine in the prevention and treatment of radiation-induced lung injury in the prior art.

Contents of the invention

The purpose of the present invention is to provide a new application of glucosamine, that is, the application in the preparation of medicines for the prevention and treatment of radiation-induced lung injury caused by ionizing radiation.

In order to achieve the above object, the first aspect of the present invention provides the application of glucosamine in the preparation of drugs for the prevention and treatment of ionizing radiation-induced radiation-induced lung injury, the structural formula of the glucosamine is shown in the following formula I:

Further, the glucosamine reduces the damage of lung tissue cells caused by ionizing radiation.

Further, the glucosamine inhibits the inflammatory response of lung tissue caused by ionizing radiation.

Further, the glucosamine inhibits the epithelial-mesenchymal transition of lung tissue induced by ionizing radiation.

Further, the glucosamine inhibits the overexpression of the interstitial markers α-SMA and Vimentin induced by ionizing radiation, and inhibits the downregulation of the interstitial marker E-cadherin induced by ionizing radiation.

Further, the drug for prevention and treatment of radiation-induced lung injury caused by ionizing radiation is an oral preparation.

Further, the dose of glucosamine in the drug for preventing and treating radiation-induced lung injury caused by ionizing radiation is 100 mg/kg/d, and it is administered 3 days before the ionizing radiation.

Further, the ionizing radiation is ⁶⁰ Co gamma ray irradiation.

The second aspect of the present invention provides a medicine for the prevention and treatment of radiation-induced lung injury, the active ingredient of which is glucosamine.

Further, the drug for preventing and treating radiation-induced lung injury by ionizing radiation also includes pharmaceutically acceptable auxiliary materials.

The present invention directly adds glucosamine of different concentrations (<15mmol/L) to normal lung epithelial cells RLE-6TN, and uses CCK-8 to detect cell growth and proliferation after 24 and 48 hours of treatment. Glucosamine did not inhibit the growth and proliferation of RLE-6TN cells.

At the same time, the present invention uses 5mmol/L glucosamine to treat RLE-6TN cells, and then irradiates the cells with different doses (0, 2, 4, 8Gy) of ⁶⁰ Co gamma rays, and then continues to culture for 2 weeks and then uses the colony formation rate to detect cell growth and proliferation, the results showed that after pretreatment with glucosamine (5mmol/L) at a concentration without toxic side effects, the cell death rate of RLE-6TN cells induced by radiation therapy could be significantly reduced with the increase of irradiation dose. At the same time, the present invention uses 5mmol/L glucosamine to treat RLE-6TN cells, then irradiates the cells with 8Gy of 60Coγ-rays, and then continues to culture for 24 hours to detect cell apoptosis by staining with Hoechst33342. The results show that the concentration of glucose without toxic and side effects Amine (5mmol/L) pretreatment can significantly reduce the apoptosis rate of RLE-6TN cells induced by radiotherapy, and the concentration of glucosamine used has no effect on RLE-6TN. These research data show that glucosamine can reduce the damage of normal lung tissue cells at concentrations without obvious side effects.

In addition, the present invention uses 8-week-old female C57BL/6 mice obtained from the Experimental Animal Center of the Chinese Academy of Sciences for animal experiments. Mice were housed in cages with daily change of litter at 25±1°C. The mice were randomly divided into 4 groups: group 1, non-irradiated + saline control group; group 2, irradiated + saline group; group 3, irradiated + WR2721 group; group 4, irradiated + glucosamine group. Glucosamine (100 mg/kg/d) and WR2721 (50 mg/kg/d) were intraperitoneally injected into the corresponding groups 3 days before partial irradiation of the whole lung. Then continue to feed the mice for 1 week and 8 weeks, then sacrifice the mice, take out the lung tissue, fix it, embed it in wax block, and perform HE staining after sectioning. The results showed that the structure of the alveolar wall in the non-irradiated group was normal. One week after irradiation, the alveolar septa were thickened and a large number of inflammatory cells infiltrated in the mice of the simple irradiation group. At 8 weeks after the irradiation, the alveolar wall thickened and aggravated, the alveolar collapsed to varying degrees, the fibroblasts proliferated significantly, and the alveolar wall showed varying degrees of fibrosis. By comparing the simple irradiation group with the glucosamine treatment group, it can be clearly found that glucosamine exhibits a good anti-inflammatory effect in the early stage of lung tissue exposure. At the same time, in the comparison of the degree of late inflammation, the glucosamine treatment group had significantly less inflammatory response. In the WR2721-treated group, the irradiated lung tissue of the mice also showed a milder inflammatory response.

At the same time, in the present invention, immunohistochemical staining of α-SMA, Vimentin and E-cadherin is carried out after the embedded wax block is sliced. The results showed that glucosamine inhibited the overexpression of the interstitial markers α-SMA and Vimentin induced by ionizing radiation, and inhibited the downregulation of the interstitial marker E-cadherin induced by ionizing radiation. The treatment effect of WR2721 was significantly weaker than that of the glucosamine group. It shows that glucosamine has a significant inhibitory effect on ionizing radiation-induced epithelial-mesenchymal transition in lung tissue.

Therefore, the present invention claims to protect the application of glucosamine in the preparation of medicines for the prevention and treatment of radiation-induced lung injury caused by ionizing radiation.

Glucosamine provided by the present invention has the following advantages as a medicine for preventing and treating radiation-induced lung injury caused by ionizing radiation:

1. Low toxicity and side effects. At present, glucosamine has been widely used in the field of joint inflammation. It has been reported in the literature that glucosamine is used as a health product in the United States, Japan and Europe;

2. The curative effect is remarkable. Administration at a dose of 100 mg/kg/d 3 days before irradiation can significantly protect the lung tissue and inhibit the epithelial-mesenchymal transition of the lung tissue. The above properties all show the uniqueness of glucosamine in the protection of ionizing radiation-induced radiation-induced lung injury, and it has broad application prospects in the medical field of our country.

Description of drawings

Fig. 1 is the effect of 24 hours (A) and 48 hours (B) of different concentrations (<15 μmol/L) of glucosamine in embodiment 1 on RLE-6TN cell growth and proliferation;

Fig. 2 is the change of growth and proliferation of RLE-6TN cells pretreated with glucosamine under different irradiation doses in Example 2 after irradiation;

Fig. 3 is the change of apoptosis of RLE-6TN cells pretreated with glucosamine in Example 3;

Fig. 4 is a comparison of HE slices after whole lung irradiation of mice pretreated with glucosamine in Example 4.

5 (A-D) are comparisons of α-SMA, Vimentin and E-cadherin immunohistochemical staining sections after whole lung irradiation of glucosamine-pretreated mice in Example 5.

Detailed ways

The specific implementation modes provided by the present invention will be described in detail below in conjunction with the examples.

Materials: cell line and cell culture: Rat normal lung epithelial cells RLE-6TN (American Cell Collection Center) were cultured in RMPI 1640 medium containing 10% fetal bovine serum in a 37°C, 5% CO ₂ incubator. Drugs and main reagents: drug glucosamine, dissolved in PBS, made into a mother solution of 1mol/L and stored in a 4-degree refrigerator. RMPI 1640 medium, fetal bovine serum, trypsin and WR2721 were purchased from Gibco; CCK-8 reagent was purchased from Tongjin Chemical Research Institute; crystal violet, Hoechst staining solution, and propidium iodide (PI) were purchased from Jiangsu Biyuntian Biology Co., Ltd. Institute of Technology. Mice: 8-week-old female C57BL/6 mice obtained from the Experimental Animal Center of the Chinese Academy of Sciences.

Example 1:

(1) Cell culture: RLE-6TN cells were cultured in RMPI medium containing 10% fetal bovine serum. All the cells were cultured in a 37°C, 5% CO ₂ incubator, passaged every 2-3 days, and the cells in the logarithmic growth phase were used for experiments.

(2) CCK-8 colorimetric method: cells in logarithmic growth phase (5*10 ⁴ /mL) were inoculated into 96-well plates 24 hours before drug treatment, and 6 parallel wells were set for each concentration. Different concentrations of glucosamine were added to continue culturing for 24 and 48 hours. Add 10 μL CCK-8 reagent to the cell culture medium and continue culturing for 1-4 hours. The absorbance at 450 nm was measured with a microplate reader. Finally, the formula of cell survival rate=dosage group value/control group value×100% was used to calculate the cell survival rate.

The results obtained are shown in Figure 1, and the results show that glucosamine less than 10mmol/L does not inhibit the growth and proliferation of RLE-6TN cells. The median lethal concentration (IC 50 ) of RLE-6TN cells treated with glucosamine for 48 hours was 14.9mmol/L.

Example 2:

(1) Cell culture is the same as in Example 1;

(2) Cell clone formation method: Take RLE-6TN cells in the logarithmic growth phase, and inoculate different numbers of cells into six-well plates according to different irradiation dose requirements (0, 2, 4, and 8 Gy doses inoculated with 200 cells, respectively. 400, 800 and 1600). After 24 hours, the glucosamine combined with radiation group was treated with 5 mmol/L glucosamine for 1 hour, and the control group was treated with the same amount of PBS as a negative control. The two groups of cells received different doses (0-8Gy) of ⁶⁰ Coγ-ray irradiation at the same time, and continued to culture for 24 hours after irradiation. Both groups were replaced with ordinary medium, and the culture continued until cell clones visible to the naked eye appeared in the culture dish. . Discard the medium, wash twice with PBS, fix with anhydrous methanol for 30 minutes, stain with Gimsa stain for 30 minutes, rinse with running water and dry in the air. Count the clonal colonies of more than 50 cells under a microscope, set at least 3 parallel samples at each measurement point, take the average value, and calculate the cell survival rate. The results obtained are shown in Figure 2. After pretreatment with glucosamine (5 mmol/L) at a concentration without toxic side effects, the cell death rate of RLE-6TN cells induced by radiotherapy can be significantly reduced with the increase of irradiation dose.

Example 3:

(1) Cell culture is the same as in Example 1;

(2) Hoechst33342 staining method to detect cell apoptosis: RLE-6TN cells (1*10 ⁵ /mL) in the logarithmic growth phase were inoculated into six-well plates overnight, treated with 5 mmol/L glucosamine, and then given 8 Gy of glucosamine to the cells. After irradiating with 60Coγ-rays and continuing to culture for 24 hours, they were washed three times with PBS and fixed with paraformaldehyde. Cell apoptosis was detected after staining with Hoechst33342 and PI. The obtained results are shown in Figure 3, the pretreatment of glucosamine (5mmol/L) with no toxic and side effect concentration can significantly reduce the apoptosis rate of RLE-6TN cells induced by radiotherapy, and the glucosamine of the concentration used has no effect on RLE-6TN cells. 6TN makes an impact.

Example 4:

(1) Feeding of mice: The mice were placed in cages with daily bedding changes at 25±1°C to ensure adequate water and food.

(2) First establish a mouse model of radiation-induced radiation-induced lung injury, select 6-8 week-old female C57BL/6 mice, and randomly divide them into three groups: the irradiation group (15Gy) with 8 mice and the glucosamine administration group 3 days before irradiation 8 rats, 8 rats in the WR2721 administration group and 8 rats in the control group; ⁶⁰ Coγ-rays were used to irradiate the chest of the mice once, and the absorbed dose of the mice was 15Gy. Glucosamine (100 mg/kg/d) and WR2721 (50 mg/kg/d) were delivered to the corresponding groups by intragastric administration. Mice were observed and recorded every morning and evening after radiation exposure. The mice were sacrificed 1 week and 8 weeks after irradiation, and the lung tissues were taken out, fixed, embedded in wax blocks, and sliced for HE staining. The results obtained are shown in Figure 4, and the alveolar wall structure of the non-irradiated group was normal. One week after irradiation, the alveolar septa were thickened and a large number of inflammatory cells infiltrated in the mice of the simple irradiation group. At 8 weeks after the irradiation, the alveolar wall thickened and aggravated, the alveolar collapsed to varying degrees, the fibroblasts proliferated significantly, and the alveolar wall showed varying degrees of fibrosis. By comparing the simple irradiation group with the glucosamine treatment group, it can be clearly found that glucosamine exhibits a good anti-inflammatory effect in the early stage of lung tissue exposure. At the same time, in the comparison of the degree of late inflammation, the glucosamine treatment group had significantly less inflammatory response. In the WR2721-treated group, the irradiated lung tissue of the mice also showed a milder inflammatory response.

Example 5:

(1) Feeding of mice: The mice were placed in cages with daily bedding changes at 25±1°C to ensure adequate water and food.

(2) The mouse model of radiation-induced radiation-induced lung injury is the same as in Example 4.

(3) The lung tissue wax blocks of each group in Example 4 were taken for immunohistochemical staining of α-SMA, Vimentin and E-cadherin. Slices were photographed and counted. The results obtained are shown in Figure 5, glucosamine inhibits the overexpression of the interstitial markers α-SMA and Vimentin induced by ionizing radiation, and inhibits the downregulation of the interstitial marker E-cadherin induced by ionizing radiation. The treatment effect of WR2721 was significantly weaker than that of the glucosamine group. It shows that glucosamine has a significant inhibitory effect on ionizing radiation-induced epithelial-mesenchymal transition in lung tissue.

Among them, irradiation conditions: ⁶⁰ Co γ-ray irradiation at the Radiation Center (Naval Medical College, Second Military Medical University, Shanghai, China). After anesthetized with 10% chloral hydrate (350 mg/kg), mice were subjected to whole lung irradiation. All irradiated animals received a single dose of 15Gy at a dose rate of 1Gy/min and were monitored for up to 1 week after irradiation. Cells were treated with 8Gy gamma-ray irradiation at a dose rate of 1Gy/min.

Statistical processing: all experiments of above-mentioned embodiment are all repeated more than 3 times, the result adopts express. SAS statistical software was used to carry out t test on relevant data, and P<0.05 was considered significant difference.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. These equivalent modifications or replacements are all included within the scope defined by the claims of the present application.

Claims (10)

1. the application of glucosamine in the preparation of ionizing radiation-induced radiation-induced lung injury prevention and treatment medicine, the structural formula of said glucosamine is as shown in formula I: 2. The application of glucosamine according to claim 1 in the preparation of medicines for the prevention and treatment of ionizing radiation-induced radiation-induced lung injury, wherein said glucosamine reduces the damage of lung tissue cells caused by ionizing radiation. 3. The application of glucosamine according to claim 1 in the preparation of medicines for the prevention and treatment of lung injury caused by ionizing radiation, characterized in that, said glucosamine inhibits the inflammatory response of lung tissue caused by ionizing radiation. 4. The application of glucosamine according to claim 1 in the preparation of medicines for preventing and treating radiation-induced lung injury caused by ionizing radiation, wherein said glucosamine inhibits the epithelial-mesenchymal transition of lung tissue induced by ionizing radiation. 5. the application of glucosamine according to claim 4 in the preparation of ionizing radiation-induced radiation-induced lung injury prevention and treatment medicine, is characterized in that, described glucosamine inhibits the excessive expression of interstitial markers α-SMA and Vimentin caused by ionizing radiation , and inhibit the down-regulation of the interstitial marker E-cadherin induced by ionizing radiation. 6. The application of glucosamine according to claim 1 in the preparation of medicines for preventing and treating lung injury caused by ionizing radiation, characterized in that the medicine for preventing and treating lung injury caused by ionizing radiation is an oral preparation. 7. the application of glucosamine according to claim 1 in the preparation of ionizing radiation-induced radiation-induced lung injury prevention and treatment medicine, is characterized in that, the dosage of glucosamine in the described ionization radiation-induced radiation-induced radiation-induced lung injury prevention and treatment medicine is 100mg/ kg/d, and administered 3 days before ionizing radiation. 8. The application of glucosamine according to claim 1 in the preparation of medicines for the prevention and treatment of ionizing radiation-induced radiation-induced lung injury, characterized in that the ionizing radiation is ⁶⁰ Co gamma ray irradiation. 9. A medicine for preventing and treating radiation-induced lung injury caused by ionizing radiation, characterized in that the active ingredient of the medicine for preventing and treating radiation-induced lung injury caused by ionizing radiation is glucosamine. 10. The medicine for preventing and treating lung injury caused by ionizing radiation according to claim 9, characterized in that the medicine for preventing and treating lung injury caused by ionizing radiation further comprises pharmaceutically acceptable excipients.