Use of isoxazepine in the preparation of a medicament for preventing or treating radiation-induced testicular damage

Abstract

The present application relates to the field of medicine, specifically is the application of isoxazoline in the preparation of drugs for preventing or treating radiotherapy testicular injury. The present application finds that isoxazoline has a protective effect on testicular injury after irradiation, and confirms that it can reduce pathological injury in the testis after irradiation, inhibit cell apoptosis, reduce DNA damage, oxidative stress level (MDA, SOD, GSH), and reduce the content of inflammatory factors (IL-1β, IL-6, IL-18, TNF-α), so that isoxazoline has a wide application prospect as a potential radiation protection agent in the development of drugs for treating radiotherapy testicular injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, the application of Isofraxidin in the preparation of a drug for preventing or treating radiation-induced testicular damage. BACKGROUND

[0002] With the development of clinical technology, radiotherapy is widely used in male tumor patients, such as testicular cancer, prostate cancer, Hodgkin's lymphoma, etc. Radiotherapy improves the treatment effect of tumors to some extent, but also increases the incidence of male infertility and sexual dysfunction. The testis is a radiosensitive tissue, and a dose of 0.35 Gy can cause temporary azoospermia, and a dose of >2 Gy can cause temporary infertility for 10-24 months, and the recovery time is prolonged with the increase of the irradiation dose, and a dose of >6 Gy can cause permanent infertility. Radiation-induced testicular damage is characterized by atrophy of the spermatogenic tubules and reduction of sperm quality, which can cause temporary or permanent infertility.

[0003] Radioprotectors are prophylactically used before radiation to reduce radiation damage. FDA-approved radioprotectors such as amifostine and filgrastim have been clinically applied to reduce organ toxicity caused by radiotherapy. However, so far there is no effective radioprotector for radiation-induced testicular damage. Therefore, reproductive damage caused by radiotherapy seriously affects the quality of life of cancer patients. It is urgent to develop new radioprotectors for intervention, which can also benefit normal tissues and cells in the clinic and improve the treatment effect of cancer.

[0004] Isofraxidin (IF) is also known as 7-hydroxy-6,8-dimethoxybenzopyran-2-one, which is a hydroxycoumarin monomer widely present in the rhizome, leaf and fruit of Eleutherococcus senticosus, and has anti-inflammatory, antioxidant and antitumor pharmacological activities.

[0005] Chinese patent document CN103623017A discloses a Ilex hainanensis extract and its application, which is prepared by water extraction, concentration and drying of Ilex hainanensis whole grass. 1 g of the extract contains not less than 0.53 mg of Isofraxidin and not less than 0.38 mg of rosmarinic acid, and has good effects on treating and preventing xerostomia and radio caries caused by radiotherapy. However, so far there is no systematic research report on Isofraxidin for preventing and treating radiation-induced testicular damage. SUMMARY

[0006] The present application aims to provide a new use of Isofraxidin, i.e. the application of Isofraxidin in the preparation of a drug for preventing or treating radiation-induced testicular damage, so as to realize effective treatment of radiation-induced testicular damage.

[0007] In order to achieve the above-mentioned purpose, the first aspect of the present application provides the application of Isofraxidin in the preparation of a drug for preventing or treating radiation-induced testicular damage.

[0008] Further, the medicine for preventing or treating the radiation-induced testicular injury is a medicine composition comprising isofraxidin as the only active monomer component or isofraxidin and at least one pharmaceutically acceptable excipient.

[0009] Further, the purity of isofraxidin in the medicine for preventing or treating the radiation-induced testicular injury is not less than 95%.

[0010] Further, the dosage of isofraxidin is 10 mg / kg-40 mg / kg, preferably 40 mg / kg.

[0011] Further, the medicine for preventing or treating the radiation-induced testicular injury is an intravenous injection.

[0012] Further, the radiation in the radiation-induced testicular injury is gamma rays.

[0013] Further, the radiation-induced testicular injury mainly includes damage to the seminiferous tubules and the surrounding connective tissue.

[0014] Further, the medicine for preventing or treating the radiation-induced testicular injury is a medicine for improving the pathological damage of the testicular tissue of mice caused by radiation, including reducing the atrophy of the seminiferous tubules and the loss of spermatocytes.

[0015] Further, the medicine for preventing or treating the radiation-induced testicular injury is a medicine for increasing the testosterone content and maintaining the hormone level in the testis of mice.

[0016] Further, the medicine for preventing or treating the radiation-induced testicular injury is a medicine for inhibiting the occurrence of tissue damage, cell apoptosis, DNA damage, inflammatory response and oxidative stress disorder in the testis caused by radiation.

[0017] The second aspect of the present application provides a medicine for preventing or treating the radiation-induced testicular injury, which is a medicine composition comprising isofraxidin as the only active monomer component or isofraxidin and at least one pharmaceutically acceptable excipient.

[0018] Isocyanidin, a representative coumarin active ingredient of Acanthopanax senticosus, has been verified to possess various biological functions, including antioxidant, anti-stress, and anti-inflammatory effects. This invention provides the application of isocyanidin in the preparation of drugs for the prevention or treatment of radiation-induced testicular injury. By establishing an animal model of radiation-induced testicular injury, the protective effect and possible mechanism of isocyanidin against irradiated testicular injury are explored. After ionizing radiation, isocyanidin can alleviate radiation-induced pathological damage in the testes, inhibit testicular cell apoptosis, reduce DNA damage, decrease the level of inflammatory factors in the testes, and regulate oxidative stress disorders, demonstrating potential application value in drugs for the prevention and treatment of radiation-induced testicular injury.

[0019] The advantages of this invention are:

[0020] This invention has discovered the protective effect of isozincide against irradiated testicular injury, confirming that it can reduce pathological damage in the testes after radiation, inhibit cell apoptosis, reduce DNA damage, decrease oxidative stress levels (MDA, SOD, GSH), and reduce the content of inflammatory factors (IL-1β, IL-6, IL-18, TNF-α). Therefore, isozincide has broad application prospects as a potential radiation protectant in the development of drugs for the treatment of radiation-induced testicular injury. Attached Figure Description

[0021] Figure 1 This is a schematic diagram showing the radiation protection effect of isozymine on mouse testes.

[0022] Figure 2 This is a schematic diagram illustrating the results of isozymidine-regulated radiation-induced oxidative stress disorder in the testes of mice.

[0023] Figure 3 This is a schematic diagram showing the antioxidant activity of isozymidine mediated by the Nrf2 / HO-1 pathway in radiation-induced testicular injury.

[0024] Figure 4 This is a schematic diagram showing the activation of inflammatory response in the testes of mice after isozymidine reduces radiation exposure.

[0025] Figure 5 This is a schematic diagram showing the anti-inflammatory activity of isozymidine mediated by the NLRP3 / ASC pathway in radiation-induced testicular injury. Detailed Implementation

[0026] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0027] In this embodiment of the invention, isozyzidine was purchased from MedChemExpress (HY-N0774). The male C57BL / 6J mice used in the experiment were 7-8 weeks old, weighed 20±2g, and were SPF-grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The room temperature was approximately 25℃, and the mice had free access to purified water and standard rodent feed. All experimental procedures and related operations were approved by the Laboratory Animal Ethics Center of the Academy of Military Medical Sciences (IACUC-DWZX-2021-557).

[0028] Experimental Example 1: Protective effect of isozymine on testicular damage in irradiated mice

[0029] 1. Experimental grouping and drug administration

[0030] Normal group: Mice were given physiological saline containing 5% DMSO; Normal + Isoprena + Irradiation group: 12 hours before irradiation, mice were intraperitoneally injected with 10 mg, 20 mg, or 40 mg / kg isoprena (dissolved in 5% DMSO physiological saline) and then irradiated with 5 Gy of cobalt-60 γ-rays. 12 hours after irradiation, mice were given isoprena in different concentrations again; Irradiation group: Mice were given physiological saline containing 5% DMSO and then irradiated with 5 Gy of cobalt-60 γ-rays.

[0031] 2. Experimental Methods

[0032] (1) Hematoxylin-eosin staining to detect pathological damage in testicular tissue

[0033] Tissue sections were soaked in xylene I and II for 30 minutes each; then soaked in ethanol of different concentrations in turn, and rinsed with distilled water for 1 minute; then stained with hematoxylin-eosin; the stained sections were mounted with neutral resin and observed under an optical microscope.

[0034] (2) Immunofluorescence assay of testicular tissue

[0035] 1) After fixing the testicular tissue with 4% paraformaldehyde for two days, it was replaced with 70% ethanol for preservation. The tissue was then trimmed into 5mm tissue blocks and placed in an embedding cassette.

[0036] 2) The tissue was dehydrated by soaking it in 70% ethanol, 85% ethanol, 90% ethanol, 95% ethanol and anhydrous ethanol for 60 minutes in sequence, then treating it with dewaxing solution I, dewaxing solution II and dewaxing solution III for 30 minutes in sequence, and then treating it with paraffin I, paraffin II and paraffin III for 60 minutes in sequence.

[0037] 3) After embedding the tissue with an embedding machine, cut the paraffin sections to 3μm, and then dewax the paraffin sections. The process is as follows: baking the sections (60℃, 120min) → soaking in dewaxing solutions for 5min each (xylene II, xylene I, alcohol + xylene, 100% alcohol II, 100% alcohol I, 95% alcohol, 90% alcohol, 85% alcohol, 70% alcohol) → washing twice with distilled water;

[0038] 4) Antigen retrieval was performed using EDTA (pH 9.0) at 121℃ (for 2 min). After natural cooling, the antigen was washed twice with PBS, then blocked sequentially with hydrogen peroxide and 1% BSA (washed twice with PBS in between), and then incubated overnight at 4℃ with primary antibody (prepared with 1% BSA).

[0039] 5) After incubating with the primary antibody overnight, wash away the primary antibody and wash three times with PBS. Then, incubate with horseradish peroxidase-labeled secondary antibody for 90 min and wash three times with PBS. Next, develop the color with 3-diaminobenzidine (DAB), then counterstain with hematoxylin and rinse to return to blue. Finally, dehydrate and mount with neutral resin.

[0040] (3) TUNEL staining was used to detect the level of cell apoptosis in the testicular tissue of mice in each group.

[0041] 1) Dewax testicular paraffin sections in xylene for 5-10 minutes. Replace with fresh xylene and dewax again for 5-10 minutes → 5 minutes in anhydrous ethanol → 2 minutes in 90% ethanol → 2 minutes in 70% ethanol → 2 minutes in distilled water.

[0042] 2) Add 20 μg / ml of DNase-free protease and incubate at 37°C for 20 minutes.

[0043] 3) Wash 3 times with PBS or HBSS.

[0044] 4) Add 50 μl of TUNEL detection solution to the sample and incubate at 37°C in the dark for 60 minutes.

[0045] 5) Wash 3 times with PBS or HBSS.

[0046] 6) After mounting with an anti-fluorescence quenching mounting solution, observe under a fluorescence microscope. The usable excitation wavelength range is 450-500 nm, and the emission wavelength range is 515-565 nm (green fluorescence).

[0047] (4) Determination of testosterone content in mouse testes by enzyme-linked immunosorbent assay (ELISA)

[0048] 1) Add 100 μl of different concentration standards and test samples to the microplate in two replicates.

[0049] 2) Seal the ELISA plate with sealing film and place it in a 37°C incubator for 90 minutes.

[0050] 3) After the reaction is complete, shake the microplate dry. Then add 100 μl of the corresponding indicator antibody working solution to each well. Repeat the pipetting and mixing process. After sealing with the sealing film, incubate at 37°C for 60 minutes.

[0051] 4) After the reaction is complete, pour out the reagents from the wells of the microplate, wash 5 times with 300 μl of washing buffer per well, and then tap out the washing solution.

[0052] 5) After washing the ELISA plate, add 90 μl of TMB chromogenic solution to each well using a pipette, and then incubate at 37°C in the dark for 20 minutes.

[0053] 6) After the reaction, remove the microplate and add 100 μl of stop solution to each well using a pipette. Immediately after adding the stop solution, place the plate into the microplate reader for detection and measure the absorbance at 450 nm.

[0054] 3. Experimental Results

[0055] like Figure 1 As shown, Figure 1 This is a schematic diagram showing the protective effect of isozymine against testicular damage in irradiated mice.

[0056] The pathological damage of testicular tissue was detected by hematoxylin-eosin staining. Figure 1 As shown in A, 40 mg / kg isozymine can significantly improve radiation-induced pathological damage to mouse testicular tissue, including reducing seminiferous tubule atrophy and spermatocyte loss.

[0057] Testosterone plays a crucial role in regulating the structure and function of male reproductive organs, and is particularly important for spermatogenesis. Impaired testosterone synthesis leads to decreased testicular weight and impaired spermatogenesis, resulting in male infertility. This study measured testosterone levels in the testes of mice in different groups using enzyme-linked immunosorbent assay (ELISA). Figure 1 As shown in Figure B, compared with the control group, the testosterone level in the testes of the irradiated mice was significantly reduced after irradiation, while the administration of different concentrations of isozymine significantly increased the testosterone content and maintained the hormone level in the testes of the mice.

[0058] The level of DNA damage in the testes of mice in each group was detected by γ-H2AX immunofluorescence staining. Figure 1 As shown in Figure C, compared with the irradiation group, the DNA damage in the testes of mice in the isozyridine-treated group was significantly reduced, suggesting that isozyridine can alleviate DNA damage in the testes of mice after irradiation.

[0059] The level of testicular cell apoptosis in each group of mice was detected by TUNEL staining. Figure 1As shown in Figure D, compared with the irradiation group, the level of intratestinal cell apoptosis in mice in the isozygote-treated group was significantly reduced, suggesting that isozygote can inhibit intratestinal cell apoptosis in irradiated mice.

[0060] Example 2: Isocyanide-modulated radiation-induced oxidative stress disorder in mouse testes

[0061] 1. Experimental grouping and drug administration (steps are the same as in Example 1)

[0062] 2. Experimental Methods

[0063] The levels of relevant oxidative stress indicators in the testes of mice in each group were determined by enzyme-linked immunosorbent assay (ELISA) (procedure same as in Example 1).

[0064] 3. Experimental Results

[0065] Figure 2 This is a schematic diagram showing the effects of isozypilidine on relevant oxidative stress indicators in the testes of mice in each group.

[0066] Studies have shown that radiation-induced increases in ROS can lead to testicular tissue damage, causing oxidative stress, which in turn leads to lipid peroxidation. Figure 2 As shown in Figure A, compared with the irradiation group, the level of SOD, an antioxidant marker, in the testes of mice in the isozygote-treated group was significantly increased.

[0067] SOD, 8-OHDG, and MDA, as markers of lipid peroxidation, are important parameters for evaluating radiation-induced testicular damage. GSH, as a major marker of oxidative stress, may show decreased levels related to reactive oxygen species generated by intracellular water electrolysis after radiation exposure. Figure 2 As shown in B, C, D, and E, compared with the irradiation group, the isozycin group significantly reduced the levels of relevant oxidative stress indicators (ROS, 8-OHDG, MDA, GSH) in the mouse testes, suggesting that isozycin has good antioxidant properties for irradiated mouse testicular damage.

[0068] Example 3: Isocyanide mediates antioxidant activity against radiation-induced testicular injury via the Nrf2 / HO-1 pathway

[0069] 1. Experimental grouping and drug administration (steps are the same as in Example 1)

[0070] 2. Experimental Methods

[0071] The expression levels of Nrf2 and HO-1 in the testes of mice in each group were determined by immunofluorescence staining (procedure as in Example 1).

[0072] 3. Experimental Results

[0073] The Nrf2 / HO-1 pathway is considered one of the main defense mechanisms against oxidative stress, regulating the expression of multiple antioxidant genes. Further research is needed to determine whether this pathway is involved in the protective effect of isozymidine against radiation-induced testicular damage. Figure 3 As can be seen from A and B, compared with the irradiation group, the isozycin group significantly increased the expression levels of Nrf2 and HO-1 in the testes of irradiated mice, suggesting that NRF2-mediated activation of the HO-1 pathway may be the key to the radioprotective effect of isozycin.

[0074] Example 4: Isocyanide reduces radiation-induced activation of testicular inflammation in mice

[0075] 1. Experimental grouping and drug administration (steps are the same as in Example 1)

[0076] 2. Experimental Methods

[0077] The levels of relevant inflammatory factors in the testes of mice in each group were determined by enzyme-linked immunosorbent assay (ELISA) (steps were the same as in Example 1).

[0078] 3. Experimental Results

[0079] Figure 4 This is a schematic diagram showing the effect of isozymidine in reducing the activation of inflammatory responses in the testes of mice after radiation exposure. Figure 4 As can be seen from A, B, C, and D, compared with the irradiation group, the isozymine administration group can significantly reduce the content of related inflammatory factors (IL-6, TNF-α, IL-1β, IL-18) in the testes of irradiated mice and alleviate the activation of inflammatory response in the testes.

[0080] Example 5: Isocyanide mediates anti-inflammatory activity against radiation-induced testicular injury via the NLRP3 / ASC pathway

[0081] 1. Experimental grouping and drug administration (steps are the same as in Example 1)

[0082] 2. Experimental Methods

[0083] Immunofluorescence staining was used to determine the expression levels of NLRP3 and ASC in the testes of mice in each group (steps were the same).

[0084] Example 1)

[0085] The levels of NLRP3 and ASC in the testes of mice in each group were determined by enzyme-linked immunosorbent assay (same procedure as in Example 1).

[0086] 3. Experimental Results

[0087] The NLRP3 inflammasome signaling pathway plays a crucial role in regulating radiation-induced injury. NLRP3, after binding to ASC, undergoes a series of autocleavage and activation processes to produce pro-inflammatory factors IL-1β and IL-18. Figure 4The results showed that isozymidine could significantly reduce the levels of IL-1β and IL-18, suggesting that its anti-inflammatory effect may be mediated through the NLRP3 / ASC pathway.

[0088] Figure 5 This is a schematic diagram showing the effects of isozymidine on related inflammatory cytokine pathways in the testes of irradiated mice. Figure 5 The results in A and B show that, compared with the irradiation group, the isozyridine administration group significantly reduced the expression levels of NLRP3 and ASC in the testes of irradiated mice, suggesting that isozyridine may mediate the protective effect against inflammatory damage in the testes of irradiated mice through the NLRP3 / ASC inflammasome pathway.

[0089] This invention provides the application of isozyridine in the preparation of drugs for the prevention or treatment of radiation-induced testicular injury. By establishing a mouse model of radiation-induced testicular injury, it was observed that the integrity of testicular tissue was impaired after irradiation, and isozyridine could alleviate radiation-induced testicular injury. Simultaneously, TUNEL and γ-H2AX immunofluorescence experiments showed that isozyridine could inhibit apoptosis and reduce DNA damage in mouse testes. Enzyme-linked immunosorbent assay (ELISA) experiments showed that isozyridine could regulate oxidative stress disorder and inhibit the activation of inflammatory responses in irradiated mouse testes. Furthermore, immunofluorescence experiments explored the mechanism of its antioxidant and anti-inflammatory activities, indicating that isozyridine may exert its protective effect against radiation-induced testicular injury in mice through the Nrf2 / HO-1 antioxidant pathway and the NLRP3 / ASC inflammasome signaling pathway.

[0090] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Use of isoxazepine in the manufacture of a medicament for the prevention or treatment of radiation testicular damage, characterized in that, The isoxazoline piperidine is administered at a dose of 10-40 mg / kg; the radiation in the radiation-induced testicular damage is gamma radiation.

2. Use of isoxazoline piperidine according to claim 1 for the manufacture of a medicament for the prevention or treatment of radiation testicular damage, characterized in that, The drug for preventing or treating radiation-induced testicular damage is a pharmaceutical composition comprising isoxazoline piperidine as the only active monomer ingredient or isoxazoline piperidine and at least one pharmaceutically acceptable excipient.

3. Use of isofagimidine according to claim 1 for the preparation of a medicament for the prevention or treatment of radiation testicular damage, characterized in that, The drug for preventing or treating radiation-induced testicular damage is an intravenous injection.

4. Use of isoxazoline piperidine according to claim 1 for the manufacture of a medicament for the prevention or treatment of radiation testicular damage, characterized in that, The radiation-induced testicular damage includes damage to the seminiferous tubules and surrounding connective tissue.

5. Use of isoxazoline piperidine according to claim 1 for the manufacture of a medicament for the prevention or treatment of radiation testicular damage, characterized in that, The drug for preventing or treating radiation-induced testicular damage is a drug for improving radiation-induced testicular histopathological damage, including reducing seminiferous tubule atrophy and spermatocyte loss.

6. Use of isoxazoline piperidine according to claim 1 for the manufacture of a medicament for the prevention or treatment of radiation testicular damage, characterized in that, The drug for preventing or treating radiation-induced testicular damage is a drug for restoring testosterone levels and maintaining hormone levels in the testis.

7. Use of isoxazoline piperidine according to claim 1 for the manufacture of a medicament for the prevention or treatment of radiation testicular damage, characterized in that, The drug for preventing or treating radiation-induced testicular damage is a drug for inhibiting radiation-induced testicular tissue damage, cell apoptosis, DNA damage, inflammatory response, and regulating radiation-induced oxidative stress disorders in the testis.

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