Use of DNA tetrahedra in the manufacture of a medicament for preventing and / or treating radiation-induced salivary gland injury

Abstract

The application provides use of DNA tetrahedron in preparation of a medicine for preventing and / or treating radioactive sialadenosis, and belongs to the field of biological medicine. The application first finds that the DNA tetrahedron can effectively improve the salivary gland secretion function of a mouse with radioactive sialadenosis, reduce the salivary gland tissue damage caused by the radioactive rays, and effectively treat the radioactive sialadenosis. The DNA tetrahedron is safe and non-toxic, the preparation method is simple, and the DNA tetrahedron has a wide application prospect in preparation of the medicine for preventing and / or treating the radioactive sialadenosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to the use of DNA tetrahedron in the preparation of a drug for preventing and / or treating radiation-induced salivary glands damage. BACKGROUND

[0002] Radiotherapy as one of the main treatment methods for head and neck malignant tumors has achieved good therapeutic effect. However, after radiotherapy for head and neck malignant tumors, some serious post-radiotherapy complications are often caused, the most prominent of which is radiation-induced salivary glands damage. Salivary glands are generally located in the superficial layer of tumor tissue or cover the surface of tumor, and salivary glands, especially parotid glands and submandibular glands containing serous acini, are sensitive to radiation. Therefore, radiation of salivary glands cannot be avoided in radiotherapy, which often causes damage to salivary glands, especially to large salivary glands such as submandibular glands and parotid glands, resulting in hypofunction of salivary glands, and then in significant reduction of salivary flow rate and symptoms such as xerostomia, intraoral mucositis, dental caries, ulcer, pain, dysphagia, etc. Long-term can cause digestive dysfunction, malnutrition and other systemic diseases, which causes great pain to patients and seriously affects the physical and mental health and quality of life of patients after operation.

[0003] Although a large number of scholars have carried out corresponding researches on the mechanism of radiation-induced salivary glands damage, a completely effective treatment and prevention method has not been found, and the exact mechanism is still unclear.

[0004] Amifostine is an organic sulfur phosphate, which is an internationally recognized broad-spectrum cell protective agent. When combined with chemotherapy / radiotherapy, it can reduce the local and systemic toxicity related to chemotherapy / radiotherapy, thereby improving the cure rate of tumors. However, amifostine has high cost and can easily cause various adverse reactions such as nausea and vomiting, hypotension, drowsiness, chill, allergic symptoms, etc., which limits its clinical application.

[0005] In order to solve the above problems, it is urgent to develop a safe and non-toxic drug which can effectively prevent and treat radiation-induced salivary glands damage. SUMMARY

[0006] The purpose of the present application is to provide a new use of DNA tetrahedron in the preparation of a drug for preventing and / or treating radiation-induced salivary glands damage.

[0007] The "DNA tetrahedron" is a tetrahedral nanostructure formed by four DNA single strands through base complementary pairing.

[0008] The present application provides the use of DNA tetrahedron in the preparation of a drug for preventing and / or treating radiation-induced salivary glands damage.

[0009] Furthermore, the radiation-induced salivary gland injury refers to salivary gland injury caused by radiotherapy.

[0010] Furthermore, the DNA tetrahedron is formed by four DNA single strands through complementary base pairing, and the sequences of the four DNA single strands are shown in SEQ ID NO. 1 to 4.

[0011] Furthermore, the method for preparing the DNA tetrahedron includes the following steps: maintaining four DNA single strands at 85–105°C for 5–15 min, and then maintaining them at 2–8°C for 10–30 min.

[0012] Furthermore, the method for preparing the DNA tetrahedron includes the following steps: maintaining four DNA single strands at 95°C for 10 min, and then maintaining them at 4°C for 20 min.

[0013] Furthermore, the molar ratio of the four DNA single strands is 1:1:1:1.

[0014] Furthermore, the drug is a formulation prepared with DNA tetrahedrons as the active ingredient and pharmaceutically acceptable excipients.

[0015] Furthermore, the formulation is an injectable formulation.

[0016] Furthermore, the concentration of DNA tetrahedra in the injectable formulation is 100–500 nM.

[0017] Furthermore, the concentration of DNA tetrahedra in the injectable formulation is 250 nM.

[0018] Furthermore, the drug is a drug that promotes the proliferation of salivary gland cells, a drug that inhibits the apoptosis of salivary gland cells, and / or a drug that reduces the inflammatory response of salivary gland cells.

[0019] Furthermore, the drug is a drug that improves salivary secretion function and / or reduces damage to salivary gland tissue.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) The DNA tetrahedron of this invention is safe and non-toxic;

[0022] 2) The DNA tetrahedron of the present invention can promote the proliferation of salivary gland cells under radiotherapy and inhibit salivary gland cell apoptosis;

[0023] 3) The DNA tetrahedron of the present invention can reduce the inflammatory response of salivary gland cells under radiotherapy, thereby alleviating salivary gland cell damage;

[0024] 4) The DNA tetrahedron of this invention can improve the salivary gland secretion function in mice with radiation-induced salivary gland damage;

[0025] 5) The DNA tetrahedron of this invention can alleviate radiation-induced damage to salivary gland tissue;

[0026] 6) The DNA tetrahedral synthesis method of the present invention is simple, has low preparation cost, and is suitable for industrial production.

[0027] In summary, this invention is the first to discover that DNA tetrahedrons have the aforementioned beneficial effects and have broad application prospects in the preparation of drugs for the prevention and / or treatment of radiation-induced salivary gland damage.

[0028] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0029] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0030] Figure 1 Schematic diagram of DNA tetrahedral synthesis.

[0031] Figure 2 Electrophoretic diagram of DNA tetrahedrons and their single strands.

[0032] Figure 3 : Particle size distribution map of DNA tetrahedral dynamic light scattering detection.

[0033] Figure 4 In vitro cell experiments: A. Cell proliferation results; BE. Cell apoptosis results; FI. Apoptosis protein expression; JM. Inflammatory cytokine expression.

[0034] Figure 5 Mouse body weight and salivary gland function detection: A. Body weight; B. Water intake; C. Salivary gland flow rate; D. Salivary secretion delay time.

[0035] Figure 6 Immunohistochemical staining: AB.PCNA staining results; CD.TUNEL staining results; EF.CD31 staining results. Detailed Implementation

[0036] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0037] Example 1: Preparation and Identification of DNA Tetrahedrons

[0038] 1. Synthesis method

[0039] Four single-stranded DNA molecules (S1, S2, S3, and S4) were added in an equimolar ratio to a 200 μl EP tube containing 96 μl of TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0). The final concentration of the four single-stranded DNA molecules was 1 μM. The reaction solution was heated to 95 °C and maintained for 10 min, then rapidly cooled to 4 °C and maintained for 20 min to obtain DNA tetrahedra, named TDNs. A schematic diagram of the synthesis is shown below. Figure 1 As shown.

[0040] Table 1. Sequences of the four DNA single strands

[0041]

[0042] 2. Identification

[0043] Polyacrylamide gel electrophoresis showed that the DNA tetrahedron was approximately 180 bp in size. Figure 2 Dynamic light scattering (DLS) showed that its particle size was approximately 10 nm. Figure 3 ).

[0044] The following experimental examples demonstrate the beneficial effects of the present invention. The DNA tetrahedrons (TDNs) used in the following experiments were prepared in Example 1.

[0045] Experiment Example 1: In vitro cell experiments

[0046] 1. Cell proliferation experiment

[0047] Submandibular gland cells with a density of 5×10 3 Cells were seeded per well in 96-well plates and cultured for 24 hours. After a single 20 Gy irradiation, the medium was replaced with serum-free medium containing different concentrations of DNA tetrahedra 24 hours later, and the cells were cultured for another 48 hours. Cell viability was assessed using the CCK-8 assay.

[0048] The results showed that compared with the irradiation-only group, the cell viability of the 125 nM and 250 nM DNA tetrahedron administration groups increased significantly. This indicates that DNA tetrahedrons promote the proliferation of radiation-damaged submandibular gland cells. Figure 4 A).

[0049] 2. Apoptosis experiment

[0050] Submandibular gland cells with a density of 1×10 6Cells were seeded per well in 6-well plates and irradiated with 20 Gy once after 24 hours. 24 hours after irradiation, the medium was replaced with serum-free medium containing 125 nM DNA tetrahedra, and the cells were cultured for another 48 hours. Cells were washed three times with PBS, digested with EDTA-free trypsin, centrifuged at 1000 rpm for 5 minutes at room temperature, and the supernatant was discarded. 400 μL of 1× Binding Buffer was added to each tube to resuspend the cells, and the flow cytometry tubes were labeled and the cells were transferred. 5 μL of Annexin V and 5 μL of PI were added to each tube, mixed well, and incubated at room temperature in the dark for 5–15 minutes. Apoptosis was analyzed by flow cytometry within 1 hour.

[0051] The results showed that the number of apoptotic cells increased in the irradiation-only group, while the number of apoptotic cells decreased in the drug-treated group compared to the irradiation-only group (Figure B). This indicates that DNA tetrahedrons can inhibit apoptosis of radiation-damaged submandibular gland cells.

[0052] 3. Expression of apoptosis proteins and inflammatory factors

[0053] Submandibular gland cells were irradiated with 20 Gy for 24 hours, then cultured for another 48 hours in serum-free medium containing 125 nM DNA tetrahedra. Total protein was extracted using a protein extraction kit. Each protein sample was mixed with loading buffer, boiled for 3 minutes, and separated by 10% SDS-PAGE electrophoresis. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and incubated overnight at 4°C with anti-caspase 3, anti-Bcl-2, anti-Bax, anti-TNF-α, anti-IL-1β, and anti-IL-6, respectively. After rewarming, the membrane was incubated with secondary antibodies at room temperature for 1 hour. Protein bands were detected using an enhanced chemiluminescence detection system, with GAPDH as the internal control protein.

[0054] The results showed that radiation upregulated caspase 3 and Bax expression and downregulated Bcl-2 expression in submandibular gland cells, while DNA tetrahedron treatment downregulated caspase 3 and Bax expression and upregulated Bcl-2 expression. This indicates that DNA tetrahedrons inhibit radiation-damaged submandibular gland cell apoptosis by regulating the Bcl-2 / Bax / Caspase-3 signaling pathway. Figure 4 C). Furthermore, radiation upregulates the expression of inflammatory cytokines TNF-α, IL-1β, and IL-6 in submandibular gland cells, while treatment with DNA tetrahedra downregulates their expression. This indicates that DNA tetrahedra can alleviate the inflammatory response of radiation-damaged submandibular gland cells, thereby reducing cell damage. Figure 4 D).

[0055] Experiment Example 2: Animal Experiment

[0056] 1. Experimental Methods

[0057] Thirty 8-week-old female Kunming mice (purchased from Chengdu Dashuo Life Science Technology Co., Ltd.) were randomly divided into three groups: Control, IR, and IR+TDNs. They were fed a normal diet and fasted for 12 hours prior to irradiation. Irradiation conditions: RS2000 biological irradiator, 20mA current, 160kV voltage, 0.3mm copper alloy filter, dose 10Gy, dose rate 1.139Gy / min, target-skin distance 60cm. Irradiation method: After successful anesthesia with 10% chloral hydrate, the mice were placed supine in lead cages with only the neck area exposed. Mice in the IR and IR+TDNs groups were placed in the radiation field sequentially for irradiation, while the Control group was only anesthetized and not irradiated. On the second day after irradiation, the Control, IR, and IR+TDNs groups were injected intravenously with 100μL PBS, 100μL PBS, and 100μL 250nM TDNs, respectively, every two days for three consecutive weeks.

[0058] Eight weeks after irradiation, the body weight, water intake, salivary gland flow rate, and salivary secretion delay time of mice in each group were measured, and mouse salivary gland tissue was collected for immunohistochemical staining (PCNA, TUNEL, CD31).

[0059] 2. Experimental Results

[0060] At week 8 post-irradiation, both the irradiation-only group (IR group) and the drug administration group (IR+TDNs group) showed significant weight loss compared to the control group (Control group). However, the weight loss in the drug administration group was less severe than that in the irradiation-only group, and the results were statistically significant. Figure 5 A). The water intake of mice in both the irradiation group and the drug administration group was significantly increased compared to the control group, while the water intake of the drug administration group was significantly decreased compared to the irradiation group. Figure 5 B). Compared with the control group, the salivary flow rate was significantly decreased in the irradiation-only group, while the salivary flow rate in the drug administration group was not significantly different from that in the control group, but significantly increased compared with the irradiation-only group. Figure 5 C). The delay time in the irradiation-only group and the drug administration group was significantly longer than that in the control group, while the delay time in the drug administration group was significantly shorter than that in the irradiation-only group. Figure 5 D). This indicates that DNA tetrahedrons can improve salivary gland secretion function in mice with radiation-induced salivary gland damage.

[0061] At week 8 post-irradiation, immunohistochemical staining of the submandibular glands showed that the PCNA-positive cell rate was significantly lower in the irradiation-only group compared to the control group, while the PCNA-positive cell rate was significantly higher in the drug-treated group than in the irradiation-only group. Figure 6 A). The number of apoptotic cells in the irradiation-only group was significantly higher than that in the control group. Although the apoptosis rate in the drug-treated group was significantly higher than that in the control group, it was significantly lower than that in the irradiation-only group, and the changes in apoptosis rate were statistically significant. Figure 6B). CD31 expression was significantly lower in the irradiation group than in the control group, while it was upregulated in the drug-treated group compared to the irradiation group. Semi-quantitative analysis of immunohistochemical results showed that microvessel density was significantly lower in the irradiation group than in the control group, while microvessel density in the drug-treated group was not significantly different from that in the control group, but significantly higher than that in the irradiation group. Figure 6 C). This indicates that DNA tetrahedrons can alleviate radiation-induced damage to salivary gland tissue.

[0062] The above results indicate that DNA tetrahedrons can effectively improve salivary gland secretion function in mice with radiation-induced salivary gland damage, reduce radiation-induced salivary gland tissue damage, and effectively treat radiation-induced salivary gland damage.

[0063] In summary, this invention is the first to discover that DNA tetrahedrons can effectively improve salivary gland secretion function in mice with radiation-induced salivary gland damage, alleviate radiation-induced salivary gland tissue damage, and effectively treat radiation-induced salivary gland damage. DNA tetrahedrons are safe and non-toxic, and their preparation method is simple, showing broad application prospects in the preparation of drugs for the prevention and / or treatment of radiation-induced salivary gland damage.

Claims

1. Use of DNA tetrahedrons in the preparation of medicaments for the prevention and / or treatment of radiation-induced salivary gland damage, wherein the radiation-induced salivary gland damage is salivary gland damage caused by radiotherapy, and the medicaments are for reducing radiation-induced salivary gland tissue damage; wherein the DNA tetrahedron is formed by four DNA single strands through complementary base pairing, and the sequences of the four DNA single strands are shown in SEQ ID NO. 1-4.

2. The use as described in claim 1, characterized in that, The method for preparing the DNA tetrahedron includes the following steps: maintaining four DNA single strands at 85-105℃ for 5-15 min, and then maintaining them at 2-8℃ for 10-30 min.

3. The use as described in claim 2, characterized in that, The method for preparing the DNA tetrahedron includes the following steps: maintaining four DNA single strands at 95°C for 10 min, and then maintaining them at 4°C for 20 min.

4. The use as described in claim 1, characterized in that, The drug is a formulation prepared with DNA tetrahedrons as the active ingredient and pharmaceutically acceptable excipients.

5. The use as described in claim 4, characterized in that, The preparation is an injectable preparation.

6. The use as described in claim 5, characterized in that, The concentration of DNA tetrahedra in the injectable formulation is 100–500 nM.

7. The use as described in claim 6, characterized in that, The concentration of DNA tetrahedra in the injectable formulation is 250 nM.

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