Application of magnolia alkaloids in the preparation of drugs for treating or preventing hearing loss

Abstract

The present invention relates to the field of biomedicine, and more particularly to the use of magnolia alkaloids in the preparation of medicaments for treating or preventing hearing loss. Experimental findings indicate that magnolia alkaloids can treat or prevent cisplatin-induced ototoxic hearing loss by inhibiting cisplatin-induced apoptosis of cochlear hair cells or reducing cisplatin-induced hair cell loss in cochlear explants. Furthermore, magnolia alkaloids are a natural herbal ingredient with no toxic effects on the cochlea, enabling patients to achieve excellent therapeutic outcomes without compromising their quality of life.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of magnolia alkaloids in the preparation of medicines for treating or preventing hearing loss. Background Art

[0002] Magnoflorine is an isoquinoline alkaloid found primarily in plants of the Magnoliaceae family, such as magnolia and its relatives. It possesses a well-defined molecular structure and complex pharmacological activities. As a natural herbal ingredient, it exhibits diverse pharmacological activities, including antioxidant, anti-inflammatory, neuroprotective, immunomodulatory, and antihypertensive properties.

[0003] Drug-induced hearing loss is a common type of hearing impairment in clinical practice. According to statistics, approximately 10%-15% of sensorineural hearing loss worldwide is caused by the use of ototoxic drugs. These drugs include a wide variety of antibiotics (such as aminoglycosides), anti-tumor drugs, and diuretics. These drugs damage the structure and function of the inner ear through various mechanisms, leading to hearing loss.

[0004] Among the many ototoxic drugs, cisplatin has become a frequently used chemotherapy drug in clinical practice due to its excellent efficacy in the treatment of various malignant tumors such as testicular cancer, ovarian cancer, and lung cancer. However, its significant side effects such as nephrotoxicity and ototoxicity have severely limited its clinical application. Currently, nephrotoxicity can be prevented and reversed early to a certain extent, but ototoxicity still lacks appropriate therapeutic and preventive means. The large-scale use of cisplatin will eventually cause bilateral, progressive, and irreversible hearing loss in patients, greatly reducing their quality of life. It is reported that cisplatin-mediated ototoxicity involves multiple mechanisms, such as oxidative stress, cell apoptosis, and inflammatory response. If a certain drug can be used to preventively protect hair cells before they are damaged to reduce the ototoxicity of cisplatin, it will have a preventive protective effect on the patient's hearing.

[0005] Currently, there is no effective treatment for cisplatin ototoxicity, and most drugs that inhibit cisplatin ototoxicity are Western medicines, which inevitably have side effects. Traditional Chinese medicine, however, has greatly increased its value in clinical drug applications due to its natural origin and lower risk of side effects. Therefore, finding a natural traditional Chinese medicine ingredient for the preventive treatment of cisplatin ototoxicity that can alleviate cisplatin toxicity without affecting anticancer efficacy is an urgent clinical challenge. Summary of the Invention

[0006] The present invention aims to provide a use of magnolamine in the preparation of a drug for treating or preventing hearing loss, so as to solve the problem that there is no effective means for preventing and treating cisplatin ototoxicity and that Western medicines for inhibiting cisplatin ototoxicity inevitably have drug side effects.

[0007] Therefore, the present invention provides the use of magnolia alkaloids in preparing a drug for treating or preventing hearing loss.

[0008] In some embodiments of the present invention, the structural formula of magnolamine is:

[0009] .

[0010] In some embodiments of the present invention, the hearing loss is drug-induced hearing loss.

[0011] In some embodiments of the present invention, drug-induced hearing loss includes cisplatin-induced ototoxic hearing loss.

[0012] In some embodiments of the present invention, the mechanism of cisplatin-induced ototoxic hearing loss is cisplatin-induced increased cochlear hair cell loss.

[0013] In some embodiments of the present invention, magnolamine treats or prevents cisplatin-induced ototoxic hearing loss by reducing the loss of cochlear hair cells caused by cisplatin and reducing or inhibiting the apoptosis of cochlear hair cells.

[0014] The present invention also provides a medicine for treating or preventing hearing loss, comprising magnolia alkaloids and a pharmaceutical carrier.

[0015] In some embodiments of the present invention, the dosage of magnolamine administered is 20-80 mg / kg.

[0016] In some embodiments of the present invention, the pharmaceutical carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers or lubricants in the pharmaceutical field.

[0017] The present invention has the following beneficial effects:

[0018] The present invention provides the use of magnolia alkaloids in the preparation of drugs for treating or preventing hearing loss. The discovery of magnolia alkaloids has changed the current situation of lack of targeted drugs for cisplatin ototoxicity, and the treatment model has changed from passive relief of ototoxicity to proactive prevention of ototoxicity.

[0019] Experimental verification has shown that magnolia alkaloids have no toxic effects on the cochlea and have fewer side effects. As a natural Chinese herbal ingredient, it is easier to obtain, has higher patient acceptance, and better compliance, allowing patients to achieve good therapeutic effects without affecting their quality of life.

[0020] The present invention also provides a drug for treating or preventing hearing loss, comprising magnolamine and a pharmaceutical carrier. The drug, which is administered orally, can provide preventive protection for hair cells through the blood-labyrinth barrier, thereby reducing the effects of chemotherapy on hearing.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing changes in ABR hearing thresholds in mice of different groups according to the present invention;

[0023] Among them, #### means p<0.0001, indicates p<0.0001, indicates p<0.001, indicates p < 0.05;

[0024] Figure 2 This is a graph showing the immunofluorescence staining results of the mouse basilar membrane hair cell marker antibody Myosin7a in the control group, cisplatin ototoxicity group, and high-dose magnolia alkaloid prevention group of the present invention;

[0025] in, Figure 2 Figure a is a schematic diagram of the staining of the top, middle and bottom sections of three groups of mouse basement membrane hair cell marker antibodies Myosin7a and cytoskeleton dye Phalloidin. Figure 2 b in the figure is a statistical diagram of the number of hair cells in the top, middle and bottom sections of the three groups. indicates p<0.0001, indicates p < 0.01;

[0026] Figure 3 : It is a diagram of hair cell staining and TUNEL apoptosis staining and statistical results of the control group, magnolia alkaloid group, cisplatin group and magnolia alkaloid pretreatment group on the explants of the present invention;

[0027] in, Figure 3 a in the figure is a diagram of the staining of the hair cell marker antibody Myosin7a and TUNEL apoptosis staining in the control group, magnolia alkaloid group, cisplatin group and magnolia alkaloid pretreatment group on the explants. Figure 3 b is the statistical results of hair cell staining and apoptosis staining in the control group, magnolia alkaloid group, cisplatin group and magnolia alkaloid pretreatment group on the explants. indicates p < 0.0001;

[0028] Figure 4 1. It is a diagram showing hair cell staining and Cleaved CASP3 staining of the control group, magnolia alkaloid group, cisplatin group and magnolia alkaloid pretreatment group on the explants of the present invention and a diagram showing statistical results;

[0029] in, Figure 4 a in the figure is a diagram of hair cell staining and Cleaved CASP3 staining in the control group, magnolia alkaloid group, cisplatin group and magnolia alkaloid pretreatment group on the explants. Figure 4 b is the statistical results of hair cell staining and apoptosis staining in the control group, magnolia alkaloid group, cisplatin group and magnolia alkaloid pretreatment group on the explants. indicates p<0.0001, Indicates p < 0.01. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to the accompanying drawings and examples. Unless otherwise defined, technical or scientific terms used herein shall have the same meanings as those commonly understood by persons of ordinary skill in the art to which the present invention pertains. The above-mentioned features or features described in the specific examples of the present invention may be combined in any manner. These specific examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] Example 1

[0032] animal

[0033] The experimental animals were 8-week-old male C57BL / 6 mice weighing 20-25 g, purchased from Jicui Yaokang.

[0034] drug

[0035] Magnolia alkaloids (purity ≥98%, purchased from Weiqi Biotechnology, product number 2141-09-5): Prepared with 0.9% saline to 80 mg / mL.

[0036] Cisplatin (cisplatin injection, purchased from Jiangsu Hausen Pharmaceutical Group Co., Ltd., batch number 601240607): diluted to 0.5 mg / mL in 0.9% saline.

[0037] Experimental groups and drug administration regimen

[0038] The 8-week-old C57BL / 6 mice were divided into a control group, a cisplatin ototoxicity group, a magnolialine treatment group, and a magnolialine prevention group. The magnolialine groups were divided into low-dose, medium-dose, and high-dose groups.

[0039] The number of mice in each group was 6 (n=6, n is the number of mice).

[0040] Control group (n=6): 8-week-old C57BL / 6 mice were fed under the same feeding conditions for 2 days. On the 3rd day after feeding, normal saline was injected into the peritoneal cavity of the mice. The injection was continued for 17 days until the 19th day, and the injection was stopped on the 20th day.

[0041] Cisplatin ototoxicity group (n=6): Under the same feeding conditions, after 8 days of feeding, the mice were treated with 3 mg / kg cisplatin by intraperitoneal injection on the 9th day for 10 consecutive days until the 19th day, and the injection was stopped on the 20th day.

[0042] Magnolia alkaloid-treated group: 8-week-old C57BL / 6 mice were fed under the same feeding conditions for two days, and then gavage-treated with 80 mg / kg magnolia alkaloid starting on the third day for 17 consecutive days until the 19th day, and gavage was stopped on the 20th day.

[0043] Magnolia alkaloid prevention group: divided into low-dose group, medium-dose group, and high-dose group;

[0044] Low-dose group (n=6): Under the same feeding conditions, after 2 days of feeding, the mice were gavaged with 20 mg / kg of magnolia alkaloids on the 3rd day. After 7 days, starting from the 9th day of feeding, the mice were gavaged with 20 mg / kg of magnolia alkaloids and then intraperitoneally injected with 3 mg / kg of cisplatin for co-treatment. The treatment lasted for 10 consecutive days until the 19th day, and gavage was stopped on the 20th day.

[0045] Medium-dose group (n=6): Under the same feeding conditions, after 2 days of feeding, the mice were gavaged with 40 mg / kg of magnolia alkaloids on the 3rd day. After 7 days, starting from the 9th day of feeding, the mice were gavaged with 40 mg / kg of magnolia alkaloids and then intraperitoneally injected with 3 mg / kg of cisplatin for co-treatment. The treatment lasted for 10 consecutive days until the 19th day, and gavage was stopped on the 20th day.

[0046] High-dose group (n=6): Under the same feeding conditions, after 2 days of feeding, the mice were gavaged with 80 mg / kg of magnolia alkaloids on the 3rd day. After 7 days, starting from the 9th day of feeding, the mice were gavaged with 80 mg / kg of magnolia alkaloids and then intraperitoneally injected with 3 mg / kg of cisplatin for co-treatment. The treatment lasted for 10 consecutive days until the 19th day, and gavage was stopped on the 20th day.

[0047] Ototoxicity Validation Method

[0048] ABR audiometry:

[0049] ABR testing was performed on mice in each group before and on the second day after drug administration. Mice were anesthetized with an intraperitoneal injection of 1% pentobarbital (10 mL / kg). The mice were placed in a soundproof box, with a recording electrode inserted subcutaneously in the midline of the brain, a reference electrode inserted behind the ear of the test ear, and a ground electrode inserted subcutaneously on the back. The mice were stimulated with short pure tone bursts using an auditory evoked potential recording system. Test frequencies were selected in sequence: 4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz, and 32 kHz. The rise and fall times were both 5 ms, and the duration was 10 ms. The critical threshold was recorded for subsequent analysis, as shown in Figure 2. Figure 1 shown.

[0050] from Figure 1As can be seen, there was no significant difference in the ABR thresholds of mice in the control group and the magnolia alkaloid-treated group, indicating that magnolia alkaloid has no side effects on mice. The threshold shifts at all frequencies of mice in the cisplatin ototoxicity group treated with cisplatin were significantly increased, while the thresholds of the high-dose magnolia alkaloid-preventive group at 4kHz, 8kHz, 12kHz, 16kHz, and 24kHz were significantly lower than those in the cisplatin ototoxicity group, with the decrease in medium and low frequencies being more significant. It can be concluded that the high-dose group, that is, the 80mg / kg dose of magnolia alkaloid pretreatment, had the most significant effect in improving cisplatin ototoxicity.

[0051] Cochlear isolation and immunofluorescence staining:

[0052] Mice in each group were killed by cervical dislocation, and their temporal bones were isolated and placed in pre-chilled phosphate-buffered saline (PBS). Excess tissue was removed under a microscope. A small hole was punctured in the cochlear apex, and a small amount of pre-chilled 4% paraformaldehyde (4% PFA, Bio-Tech) was injected into the round and oval windows of the cochlea using a syringe. The cochlea was placed in a 2 mL EP tube containing pre-chilled 4% PFA and fixed overnight in a shaking incubator at 4°C. The cochlea was then decalcified in 10% EDTA for 4-5 hours. The decalcified cochlea was placed in a dissecting dish filled with PBS, and the apical, middle, and basal turns of the cochlea were dissected under a microscope. Excess tissue, including the tectorial membrane and vestibular membrane, was removed.

[0053] Immunofluorescence staining of the mouse basilar membrane hair cell marker antibody Myosin7a in the cisplatin ototoxicity group, the control group and the high-dose magnolia alkaloid prevention group was performed and observed under a laser confocal microscope. Figure 2 As shown. Figure 2 It can be seen that the cisplatin ototoxic group showed significant loss of outer hair cells, with the most severe loss of basal outer hair cells. After pretreatment with magnolia alkaloids, the mice in the high-dose group of the magnolia alkaloid prevention group showed a significant reduction in the loss of apical, mid-, and basal outer hair cells, demonstrating a significant preventive protective effect.

[0054] Explant Verification:

[0055] Explant culture: Mice were taken for explant experiments at 3 days of age and randomly divided into 4 groups: control group, magnolialine group, cisplatin group, and magnolialine pretreatment group, with 3 mice in each group. C57BL / 6 mice were decapitated at 3 days of age, and their heads were quickly placed in pre-chilled PBS buffer. The skull was cut open along the midline of the skull at the foramen magnum, and the temporal bone was removed. Excess brain tissue and bone were removed. The intact cochlear basement membrane was isolated under a microscope and placed on a 4-well chamber slide pre-treated with Cell-Tak (Corning) and cultured for 24 hours. The culture medium consisted of DMEM / F12 medium containing B27 and N2 growth factors and 0.2% primary cell antibiotics. The culture environment was a sterile incubator at 37°C and 5% carbon dioxide.

[0056] Those cultured for 24 h without any treatment were designated as the control group (n = 3);

[0057] After 24 h of culture, the cells were treated with 80 μM magnolamine for 6 h, which was designated as the magnolamine group (n = 3);

[0058] After 24 h of incubation, the cells were treated with 50 μM cisplatin for 24 h, designated as the cisplatin group ( n = 3);

[0059] After 24 h of culture, the cells were pretreated with 80 μM magnolamine. 6 h later, the cells were co-treated with 80 μM magnolamine and 50 μM cisplatin for 24 h, which was designated as the magnolamine pretreatment group (n=3).

[0060] The basement membrane of the explants of the magnolidine group and the cisplatin group was immunostained with a hair cell marker antibody (Myosin7a), and the cell apoptosis of the isolated cochlear tissues of different groups was detected by TUNEL method. Figure 3 As shown. Figure 3 It can be seen that there is no green apoptosis fluorescence signal in the magnolia alkaloid group, indicating that magnolia alkaloid does not damage hair cells. Compared with the control group, the cisplatin group showed dense green apoptosis fluorescence signals on the explants ( p<0.0001). Compared with the cisplatin group, the green apoptosis fluorescence signal in the magnolia alkaloid pretreatment group was significantly weakened ( p<0.0001), indicating that magnolamine pretreatment can alleviate the loss of cochlear explant hair cells caused by cisplatin and alleviate the cell apoptosis induced by cisplatin treatment to a certain extent.

[0061] Immunofluorescence staining of different groups was performed using Cleaved Caspase-3 antibody. The results are shown in Figure 2. Figure 4 As shown. Figure 4It can be seen that the magnolia alkaloid group treated with magnolia alkaloid alone did not show red apoptosis fluorescence signals, indicating that magnolia alkaloid had no damaging effect on hair cells. Compared with the control group, the red apoptosis fluorescence signal expression in the cisplatin group was significantly increased ( p<0.0001), while the red apoptosis fluorescence signal in the magnolidine pretreatment group was significantly decreased compared with the cisplatin group ( p<0.01). Therefore, it can be concluded that magnolia alkaloid pretreatment can inhibit cell apoptosis to a certain extent and thus alleviate cisplatin ototoxicity.

[0062] In summary, magnolamine can treat or prevent cisplatin-induced ototoxic hearing loss by inhibiting cisplatin-induced cochlear hair cell apoptosis or by alleviating cisplatin-induced hair cell loss in cochlear explants.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The use of magnololamine in the preparation of drugs for treating or preventing hearing loss; Hearing loss is drug-induced hearing loss; Drug-induced hearing loss includes cisplatin-induced ototoxic hearing loss; The mechanism of cisplatin-induced ototoxic hearing loss is cisplatin-induced increased cochlear hair cell loss.

2. Use of the magnololamine according to claim 1 in the preparation of a drug for treating or preventing hearing loss, characterized in that: Magnolia alkaloids can treat or prevent cisplatin-induced ototoxic hearing loss by inhibiting cisplatin-induced apoptosis of cochlear hair cells or by alleviating cisplatin-induced loss of cochlear hair cells in explants.

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