Application of lupane derivative in preparation of medicine for preventing and / or treating drug-induced deafness

Abstract

The invention discloses application of lupane derivatives in preparation of medicines for preventing and / or treating drug-induced deafness, and belongs to the field of biological medicines. The lupane derivative is represented by Chiisanoside, and experiments prove that the lupane derivative can activate the expression of Dock1 and PIP5K1A, inhibit the expression of Gelsolin and inhibit hair cytoskeleton actin instability induced by ototoxic drugs, and the lupane derivative has the advantages that the lupane derivative can be used for inhibiting the ototoxic drug-induced hair cytoskeleton actin instability, and the lupane derivative can be used for inhibiting the ototoxic drug-induced hair cytoskeleton actin instability; in addition, by activating expression of TGFBR2, SLC7A11 and GPX4 at the same time, hair cell ferroptosis induced by ototoxic drugs can be inhibited; further, the effects of preventing, treating or relieving the drug-induced deafness are exerted. The lupane derivative disclosed by the invention can be further developed into a multi-target therapeutic drug for preventing, treating and relieving drug-induced deafness.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to the use of lupetane derivatives in preparing medicines for preventing and / or treating drug-induced deafness. Background Art

[0002] Drug-induced deafness refers to damage to the end organs of the eighth cranial nerve, namely the cochlea and vestibule, after or during the treatment of certain drugs or when the human body comes into contact with certain chemical reagents, which leads to sensorineural deafness and / or vestibular dysfunction, also known as ototoxicity, or drug-induced deafness for short. Due to the widespread use of antibiotics and other chemical drugs, the number of patients with drug-induced deafness is increasing day by day, making it the main cause of auditory nerve diseases. More than 100 ototoxic drugs have been found, mainly including aminoglycoside antibiotics, antimalarial drugs, analgesics, diuretics, anesthetics, anticonvulsants, anti-inflammatory drugs, anticancer drugs, anti-tuberculosis drugs, cardiovascular drugs, contraceptives, arsenic, mercury and other products. According to a survey of 2,407 acquired deaf-mute people in a district of Shanghai, 851 cases were caused by drugs, accounting for 35.4%. Among them, sensorineural deafness accounted for the majority. Sensorineural hearing loss is caused by damage to the cochlear auditory cells, auditory nerves, or neurons at all levels of the auditory conduction pathway, resulting in impaired sound perception or nerve conduction. The use of ototoxic drugs is a common cause of sensorineural hearing loss. Once ototoxicity occurs, it will cause permanent hearing loss, bring a great psychological burden to the patient, and seriously affect the patient's quality of life.

[0003] At present, the treatment and rehabilitation of drug-induced deafness is still a clinical problem. Prevention is the key to ear drug poisoning. The indications of various ear poisoning drugs should be strictly controlled to prevent abuse. Aminoglycoside antibiotics should be avoided for patients with a family history of poisoning and nephritis. Ototoxic drugs should be avoided for infants, the elderly, pregnant women, and patients with sensorineural deafness. Avoid the combined use of diuretics, anticancer drugs and aminoglycoside antibiotics. When using ototoxic antibiotics to treat diseases, regularly check hearing, and stop the drug immediately once the condition permits. Treatment should be carried out early, mainly with neurotrophic drugs, such as vitamin A, vitamin B complex, ATP, coenzyme A, hyperbaric oxygen, etc. Chinese medicine Atractylodes, Rehmannia, Lycium barbarum, etc. also have a certain protective effect on the inner ear. For those with mild poisoning in the early stage, hearing can be restored. For deafness with a long poisoning time, general treatment methods have no obvious effect. Appropriate hearing aids or electronic cochlear implants can be selected for auditory and language rehabilitation. Summary of the invention

[0004] The purpose of the present invention is to provide the use of lupine derivatives in the preparation of drugs for preventing and / or treating drug-induced deafness, so as to solve the problems existing in the above-mentioned prior art. Experiments have proved that lupine derivatives can treat drug-induced deafness induced by various drugs, indicating that lupine derivatives can be used as multi-target therapeutic drugs for preventing, treating and alleviating drug-induced deafness.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of lupine derivatives in the preparation of drugs for preventing and / or treating drug-induced deafness, wherein the lupine derivatives have a general structure as shown in Formula I:

[0007]

[0008] wherein R1 is selected from -H, -β-D-glucose, -β-D-glucose-(6←1)-β-D-glucose or -β-D-glucose-(6←1)-β-D-glucose-(6←1)-α-L-rhamnose;

[0009] R2 is selected from -H, -OH or -O(CH2)2CH3;

[0010] R3 is selected from -CH3 or -CH2OH;

[0011] R4 is selected from -OH or -H;

[0012] R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

[0013] The present invention also provides the use of lupine derivatives in the preparation of drugs for inhibiting the instability of hair cell skeleton actin induced by ototoxic drugs, wherein the lupine derivatives have a general structure shown in Formula I:

[0014]

[0015] wherein R1 is selected from -H, -β-D-glucose, -β-D-glucose-(6←1)-β-D-glucose or -β-D-glucose-(6←1)-β-D-glucose-(6←1)-α-L-rhamnose;

[0016] R2 is selected from -H, -OH or -O(CH2)2CH3;

[0017] R3 is selected from -CH3 or -CH2OH;

[0018] R4 is selected from -OH or -H;

[0019] R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

[0020] The present invention also provides the use of lupine derivatives in the preparation of drugs for inhibiting hair cell ferroptosis induced by ototoxic drugs, wherein the lupine derivatives have a general structure shown in Formula I:

[0021]

[0022] wherein R1 is selected from -H, -β-D-glucose, -β-D-glucose-(6←1)-β-D-glucose or -β-D-glucose-(6←1)-β-D-glucose-(6←1)-α-L-rhamnose;

[0023] R2 is selected from -H, -OH or -O(CH2)2CH3;

[0024] R3 is selected from -CH3 or -CH2OH;

[0025] R4 is selected from -OH or -H;

[0026] R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

[0027] Preferably, the lupeane derivative includes Chiisanoside, Chiisanogenin, Divaroside, Sessiloside A1, Elesesterpene F, Elesesterpene G, Acanthosessilioside F, Acanthosessilioside K, 1-Deoxychiisanoside, 24-Hydroxychiisanoside, 22α-Hydroxychiisanoside and 22α-Hydroxychiisanogenin. More preferably, the lupeane derivative is Chiisanoside.

[0028] Preferably, the drug-induced deafness includes cisplatin-induced deafness.

[0029] The present invention also provides a pharmaceutical composition, comprising a lupetane derivative having a chemical structure shown in the following formula I, and a pharmaceutically acceptable excipient;

[0030]

[0031] wherein R1 is selected from -H, -β-D-glucose, -β-D-glucose-(6←1)-β-D-glucose or -β-D-glucose-(6←1)-β-D-glucose-(6←1)-α-L-rhamnose;

[0032] R2 is selected from -H, -OH or -O(CH2)2CH3;

[0033] R3 is selected from -CH3 or -CH2OH;

[0034] R4 is selected from -OH or -H;

[0035] R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

[0036] The excipients are selected from any one or more of the following materials: protein, folic acid, antibody and nanomaterial; when the pharmaceutical composition is a tablet, it also includes any one or more of the following materials: binder, filler, disintegrant and lubricant; binder includes one or more of starch, methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose and sodium carboxymethyl cellulose; filler includes one or more of starch, lactose, dextrin and sucrose; disintegrant includes one or more of dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone and effervescent disintegrant; lubricant includes one or more of stearic acid, hydroxypropyl methylcellulose, polyethylene glycol and magnesium hydroxide; when the pharmaceutical composition is a pill, it also includes any one or more of the following materials: wine, vinegar and medicinal juice; when the pharmaceutical composition is a granule, it also includes the following Any one or more of the materials: filler, binder, wetting agent, disintegrant, lubricant; filler includes one or more of starch, lactose, dextrin, powdered sugar, calcium sulfate, sucrose, mannitol, microcrystalline cellulose and glucose; binder includes one or more of starch slurry, dextrin, pregelatinized starch, povidone, ethyl cellulose and hydroxypropyl cellulose; wetting agent includes distilled water or ethanol; disintegrant includes one or more of starch, sodium carboxymethyl starch, microcrystalline cellulose, cross-linked sodium carboxymethyl cellulose, low-substituted-hydroxypropyl cellulose, citric acid, polysorbate, etc.; lubricant includes one or more of stearic acid, calcium stearate, magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, sodium lauryl sulfate, paraffin, glycerol, etc.; when the pharmaceutical composition is a capsule, it also includes any one or more of the following materials: filler, lubricant, dispersant, coating agent.The filler includes one or more of starch, lactose, alumina, calcium stearate, etc.; the lubricant includes one or more of magnesium stearate, polyethylene glycol, polysorbate stearate, etc.; the dispersant includes one or more of inulin, sodium carboxymethyl cellulose, glycerol, etc.; the coating agent includes one or more of hydroxypropyl methylcellulose, gelatin, etc.; when the pharmaceutical composition is an ear drop, it also includes any one or more of the following materials: methylparaben, ethylparaben, polysorbate 80, disodium edetate, water for injection, etc.; when the pharmaceutical composition is an inhalant, it also includes any one or more of the following materials: lactose, microcrystalline cellulose, polyethylene glycol and magnesium stearate, etc.; when the pharmaceutical composition is a semisolid preparation, it also includes a matrix; when the pharmaceutical composition is a suspension injection, it also includes any one or more of the following materials: preservatives, antioxidants, flavoring agents, aromatics, solubilizers, emulsifiers , solubilizers, osmotic pressure regulators and colorants; preservatives include one or more of parabens, benzoic acid and its salts, sorbic acid, benzalkonium bromide, chloroethidine acetate, etc.; flavoring agents: divided into one or more of sweeteners, fragrances, aromatics, effervescent agents, etc.; colorants include natural pigments and synthetic pigments; antioxidants include one or more of ferrous sulfate, benzoic acid, sodium bisulfite, ascorbic acid, acetylcysteine, etc.; co-solvents include one or more of carboxymethyl cellulose, gelatin, pectin, etc.; solubilizers or emulsifiers include one or more of polyoxyethylene castor oil, polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 80 (Tween 80), povidone, lecithin, sodium deoxycholate, Pluronic F-68 (Poloxamer 188), etc.; osmotic pressure regulators include one or more of sodium chloride, glucose, glycerol, etc.

[0037] Preferably, the dosage form of the pharmaceutical composition includes but is not limited to pills, suspension injections, tablets, granules, capsules, drops or inhalants.

[0038] Preferably, the lupetane derivatives include Chiisanoside, Chiisanogenin, Divaroside, Sessiloside A1, Elesesterpene F, Elesesterpene G, Acanthosessilioside F, Acanthosessilioside K, 1-Deoxychiisanoside, 24-Hydroxychiisanoside, 22α-Hydroxychiisanoside and 22α-Hydroxychiisanogenin.

[0039] Preferably, the lupane derivative is chiisanoside.

[0040] The present invention also provides the use of the pharmaceutical composition in preparing a drug for preventing and / or treating drug-induced deafness, wherein the drug-induced deafness includes deafness induced by cisplatin.

[0041] The present invention discloses the following technical effects:

[0042] The lupetane derivatives provided by the present invention are all natural products with abundant sources. The natural products involved in the present invention are rich in the original plants (Acanthopanax senticosus and Acanthopanax breviscapus) or can be obtained by simple transformation, with safe operation and low production cost.

[0043] The present invention proves through pharmacological experiments that the lupine derivatives provided by the present invention act through precise multi-targets, convert PI4P into PIP2 by activating Dock1 / PIP5K1A protein, and then inhibit the activation of Gelsolin protein, thereby protecting HEI-OC1 cells from irreversible damage caused by ototoxic drugs-induced actin depolymerization. At the same time, ferroptosis is inhibited by activating the TGFBR2 / SLC7A11 / GPX4 axis, thereby blocking the damage to cells in the cochlea induced by ototoxic drugs. Its mechanism of action is clear, it can accurately target, make the effect more direct and effective, and can effectively reduce the generation of toxic side effects. Compared with existing clinical therapeutic drugs, the lupine derivatives provided by the present invention have both precise target correspondence and a multi-target overall intervention effect on the disease. The present invention provides reference data and new research and development ideas for the treatment of clinical drug-induced deafness. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a statistical diagram of the activity detection of HEI-OC1 cells under the action of different lupine derivatives;

[0045] Figure 2 This is a statistical chart showing the effects of CSS and its analogs on the cell viability of HEI-OC1 cells damaged by cisplatin;

[0046] Figure 3 Crystal violet staining test results for different experimental groups;

[0047] Figure 4 The effects of cell apoptosis in different experimental groups;

[0048] Figure 5 It is the pathway enrichment map of differentially expressed genes in transcriptome sequencing and the volcano map of differentially expressed genes in transcriptome sequencing;

[0049] Figure 6 This is a statistical diagram of the effects on the gene expressions of Dock1, Gelsolin, and PIP5K1A in different experimental groups;

[0050] Figure 7It is a statistical diagram of the effects on the expression of GPX4, SLC7A11, and TGFBR2 genes in different experimental groups;

[0051] Figure 8 The results of cytoskeleton actin staining in different experimental groups;

[0052] Fig. 9 It is a statistical graph of the test results of actin polymerization kit in different experimental groups;

[0053] Fig.10 Detection of DOCK1, Gelsolin, and PIP5K1A protein levels in different experimental groups;

[0054] Fig.11 It is a statistical chart of the detection results of reactive oxygen species (ROS) in different experimental groups;

[0055] Fig.12 For related biochemical index kits (GSH-PX, MDA, 4-HNE, Fe 2+ ) Test results;

[0056] Fig.13 The test results of related biochemical index kits (PI4P, PIP2, HMBG-1, LDH);

[0057] Fig.14 To detect the protein contents of GPX4, SLC7A11, and TGFBR2 in different experimental groups;

[0058] Fig.15 This is the ABR hearing threshold diagram of mice under the action of different lupine derivatives;

[0059] Fig.16 This is a statistical chart of the detection results of DOCK1 and Gelsolin protein expression in different experimental groups;

[0060] Fig.17 It is a statistical chart of the detection results of PIP5K1A and GPX4 protein expression in different experimental groups;

[0061] Fig.18 This is a statistical chart of the detection results of SLC7A11 and TGFBR2 protein expression in different experimental groups. DETAILED DESCRIPTION

[0062] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0063] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0064] Description of some terms involved in the present invention: SLC7A11: solute carrier family 7 member 11; GPX4: glutathione peroxidase 4; TGFBR2: transforming growth factor; Dock1: cytokinesis factor 1; Gelsolin: platelet skeleton protein gelsolin.

[0065] The parent core structural formula of the lupine derivatives referred to in the present invention is as follows:

[0066]

[0067] wherein R1 is selected from -H, -β-D-Glc, -β-D-Glc-(6←1)-β-D-Glc or -β-D-Glc-(6←1)-β-D-Glc-(6←1)-α-L-Rha;

[0068] R2 is selected from -H, -OH or -O(CH2)2CH3;

[0069] R3 is selected from -CH3 or -CH2OH;

[0070] R4 is selected from -OH or -H;

[0071] R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

[0072] The monomer compounds used in the following examples were extracted from the leaves (or fruits) of Acanthopanax senticosus (or Acanthopanax brevis), and their structures and content information are shown in Table 1. The extraction and separation process is briefly described as follows:

[0073] 1. Short-stemmed Acanthopanax leaves as raw material

[0074] Extraction and enrichment of total saponins: 5 kg of Acanthopanax brevis leaves (collected in mid-July, withered and dried) were ultrasonically extracted three times with 10 times the amount (volume-to-mass ratio) of 70% ethanol and water. The extracts were combined, and the ethanol was recovered until there was no alcohol taste. The mixture was suspended on a D101 macroporous adsorption resin column, and the mixture was washed with water until the effluent was colorless. The effluent was eluted with 30% ethanol until the effluent was colorless. The effluent was eluted with 50% ethanol and water until the effluent was colorless, and the eluate was collected and dissolved to obtain a 50% ethanol elution component (for standby use). The effluent was eluted with 90% ethanol and water until the effluent was colorless, and the eluate was collected and dissolved to obtain a 90% ethanol elution component (for standby use).

[0075] Separation of components eluted with 50% ethanol: take the components eluted with 50% ethanol, dry-load them onto a silica gel column (sample loading amount - silica gel = 1:20), elute with chloroform-methanol (20:1), and monitor on silica gel thin layer chromatography until no fraction is eluted; elute with chloroform-methanol (5:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain component Y-Fr-5-1; elute with chloroform-methanol (3:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain component Y-Fr-3-1; elute with chloroform-methanol (1:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain component Y-Fr-1-1.

[0076] Y-Fr-5-1 component separation: Y-Fr-5-1 components were dissolved in methanol, separated by C18 reverse phase HPLC, detected at 205 nm, and gradient eluted with acetonitrile-water (18:82-32:68). The fractions at the corresponding peaks were collected according to the chromatogram, the solvent was recovered, and the main component Chiisanoside was obtained by recrystallization with methanol.

[0077] Y-Fr-3-1 component separation: Y-Fr-3-1 components were dissolved in methanol, separated by C18 reverse phase high performance liquid chromatography, detected at 205nm, and gradient eluted with acetonitrile-water (24:76-38:62). The fractions at the corresponding peaks were collected according to the chromatogram, and the solvents were recovered respectively. The main components Divaroside and Sessiloside A1 were obtained by recrystallization with methanol.

[0078] Y-Fr-1-1 component separation: Y-Fr-1-1 component was dissolved in methanol, separated by C18 reverse phase HPLC, detected at 205nm, and gradient eluted with acetonitrile-water (33:67-41:59). The fractions at the corresponding peaks were collected according to the chromatogram, the solvent was recovered, and the main component Chiisanogenin was obtained by recrystallization with methanol.

[0079] 2. Acanthopanax senticosus fruit as raw material

[0080] Extraction and enrichment of total saponins: 5 kg of Acanthopanax senticosus fruit (collected at the end of September and dried) was ultrasonically extracted three times with 10 times the amount (volume-to-mass ratio) of 70% ethanol water. The extracts were combined, the ethanol was recovered until there was no alcohol taste, and the mixture was suspended on a D101 macroporous adsorption resin column. The mixture was washed with water until the effluent was colorless, eluted with 30% ethanol until the effluent was colorless, eluted with 50% ethanol water until the effluent was colorless, and the eluate was collected and dissolved to obtain a 50% ethanol elution component (for standby use), and eluted with 90% ethanol water until the effluent was colorless, and the eluate was collected and dissolved to obtain a 90% ethanol elution component (for standby use).

[0081] Separation of components eluted with 50% ethanol: take the components eluted with 50% ethanol, dry-load them onto a silica gel column (sample loading amount - silica gel = 1:20), elute with chloroform-methanol (20:1), and monitor on silica gel thin layer chromatography until no fraction is eluted; elute with chloroform-methanol (5:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain component G-Fr-5-1; elute with chloroform-methanol (3:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain component G-Fr-3-1; elute with chloroform-methanol (1:1), monitor on silica gel thin layer chromatography until no fraction is eluted, and recover the solvent to obtain component G-Fr-1-1.

[0082] G-Fr-5-1 component separation: G-Fr-5-1 components were dissolved in methanol, separated by C18 reverse phase HPLC, detected at 205 nm, and gradient eluted with acetonitrile-water (18:82-32:68). The fractions at the corresponding peaks were collected according to the chromatogram, and the solvents were recovered respectively. The main components 24-Hydroxychiisanoside, 22α-Hydroxychiisanoside, Acanthosessilioside K, Chiisanoside, and 1-Deoxychiisanoside were obtained by recrystallization with methanol.

[0083] G-Fr-3-1 component separation: G-Fr-3-1 components were dissolved in methanol, separated by C18 reverse phase high performance liquid chromatography, detected at 205 nm, and gradient eluted with acetonitrile-water (24:76-38:62). The fractions at the corresponding peaks were collected according to the chromatogram, the solvent was recovered, and the main component Acanthosessilioside F was obtained by recrystallization with methanol.

[0084] G-Fr-1-1 component separation: G-Fr-1-1 components were dissolved in methanol, separated by C18 reverse phase HPLC, detected at 205 nm, and gradient eluted with acetonitrile-water (33:67-41:59). The fractions at the corresponding peaks were collected according to the chromatogram, and the solvents were recovered respectively. The main components, Elesesterpene F, 22α-Hydroxychiisanogenin, Elesesterpene G, and Chiisanogenin, were obtained by recrystallization with methanol.

[0085] The structures and content information of the above separated compounds are shown in Table 1.

[0086] Table 1 Structure and content information of each monomer compound

[0087]

[0088] The structure of the above isolated compound was determined by NMR and MS data analysis. The data used for structural identification are as follows:

[0089] Chiisanoside, white crystals, C 48 H 74 O 19 . MS: [M] + 954.4815. 13 C NMR (100 MHz,MeOD) δppm: 175.00, 173.84, 150.74, 147.92, 114.60, 111.56, 104.02, 101.72, 94.89, 78.05, 77 .81, 77.68, 76.71, 76.40, 75.35, 74.93, 73.59, 73.11, 71.83, 71.83, 71.12, 70.67, 69.83 , 69.15, 61.26, 57.00, 49.84, 49.57, 47.74, 44.48, 44.25, 42.54, 41.95, 39.03, 36.98, 35.39, 33.68, 32.73, 32.20, 31.07, 29.71, 25.64, 24.71, 19.85, 19.53, 18.87, 18.38, 14.46.

[0090] Divaroside, white crystals, C 42 H 64 O 15 . MS: [M] + 808.4268. 13 C NMR (100 MHz, MeOD) δppm: 175.59, 175.56, 150.46, 147.53, 113.65, 110.23, 104.02, 94.89, 78 .12, 77.81, 77.33, 76.71, 76.42, 73.58, 73.41, 73.11, 71.12, 70.63, 69.15, 62. 26, 57.12, 50.20, 50.09, 47.76, 44.26, 44.17, 42.40, 42.20, 38.03, 36.73, 35.61, 33.56, 32.64, 32.11, 30.87, 29.63, 25.35, 22.60, 18.70, 18.51, 17.52, 13.37.

[0091] Sessiloside A1, white crystals, C 36 H 54 O 10 . MS: [M] + 646.3728. 13C NMR (100 MHz,MeOD) δppm: 175.00, 173.05, 150.16, 147.75, 113.93, 110.76, 95.55, 79.56, 78.95, 75.30, 74.36, 71.11, 70.54, 62.20, 56.81, 49.78, 49.62, 47.65, 44.15, 44.12, 42.23, 41.78, 38.83, 36.73, 35.27, 33.53, 32.39, 32.17, 30.82, 29.58, 25.22, 23.56, 19.05, 18.95, 17.91, 13.83.

[0092] Chiisanogenin, white crystals, C 30 H 44 O5. MS: [M] + 484.3169. 13 C NMR (100 MHz, CDCl3) δppm: 180.14, 172.62, 148.53, 146.35, 113.71, 110.52, 75.20, 70.69, 55.70, 49.67, 48.74, 46.63, 43.42, 43.34, 41.47, 41.08, 37.10, 36.35, 34.67, 32.08, 31.64, 31.50, 29.98, 28.79, 24.16, 22.08, 18.53, 18.46, 17.47, 13.37.

[0093] 24-Hydroxychiisanoside, white crystals, C 48 H 74 O 20 . MS: [M] + 970.4753. 13C NMR (100 MHz, Pyridine-d5) δppm: 175.11, 172.98, 152.71, 150.06, 111.35, 110.76, 105.02, 102.62, 95.38, 78.81, 78 .45, 77.98, 77.71, 76.40, 75.42, 75.23, 74.13, 73.91, 72.73, 72.53, 70.92, 70.34, 70.12 , 69.35, 67.26, 61.26, 56.81, 49.62, 47.55, 45.68, 44.33, 44.31, 42.23, 41.76, 38.36, 36.78, 35.21, 33.45, 32.83, 32.15, 30.68, 29.61, 26.71, 18.92, 18.73, 18.47, 18.12, and 13.83.

[0094] 22α-Hydroxychiisanoside, white powder, C 48 H 74 O 20 . MS: [M] + 970.4788. 13 C NMR (100 MHz, Pyridine-d5) δppm: 175.11, 173.15, 150.06, 147.68, 113.51, 110.76, 105.02, 102.62, 95.38, 78.81, 78 .45, 77.98, 77.71, 76.40, 75.42, 75.42, 75.23, 74.13, 73.91, 72.73, 72.53, 70.92, 70.34 , 70.12, 69.35, 62.51, 61.26, 49.65, 47.55, 44.33, 44.31, 44.12, 42.23, 41.65, 41.36, 38.02, 35.21, 33.45, 32.47, 29.61, 26.72, 25.24, 23.15, 19.27, 18.92, 18.47, 18.12, and 13.83.

[0095] 22α-Hydroxychiisanogenin, white powder, C 30 H 44 O6. MS: [M] + 500.3126. 13C NMR (100 MHz, Pyridine-d5) δppm: 178.52, 173.08, 151.04, 147.75, 113.86, 110.93, 75.61, 75.33, 70.58, 62.53, 49.58, 48.21, 44.35, 44.18, 44.05, 42.17, 41.77, 41.35, 38.79, 35.06, 33.75, 32.59, 29.15, 27.12, 25.37, 23.51, 19.28, 18.82, 17.34, 13.71.

[0096] 1-Deoxychiisanoside, white powder, C 48 H 74 O 18 . MS: [M] + 938.4875. 13 C NMR (100MHz, Pyridine-d5) δppm: 175.45, 175.11, 150.06, 147.65, 114.32, 110.76, 105.02, 102.62, 95.38, 78.81, 78 .45, 77.98, 77.71, 76.40, 75.42, 75.23, 74.13, 73.91, 72.73, 72.53, 70.34, 70.12, 69.35 , 61.26, 56.81, 56.81, 52.45, 49.62, 47.55, 42.23, 41.76, 39.98, 39.51, 36.78, 35.21, 33.45, 32.83, 32.15, 30.68, 30.28, 29.61, 25.33, 23.67, 18.92, 18.73, 18.47, 17.62, 13.83.

[0097] Elesesterpene F, white powder, C 30 H 44 O7. MS: [M] + 532.3059. 13C NMR (100 MHz, Pyridine-d5) δppm: 178.52, 178.02, 173.08, 147.75, 113.86, 75.33, 70.58, 56.88, 49.58, 48.21, 44.35, 44.18, 44.05, 42.91, 4 2.17, 41.77, 38.79, 37.12, 35.12, 35.06, 32.65, 32.59, 29.15, 25.78, 25.37, 23.51, 18.82, 18.75, 17.34, 13.71.

[0098] Acanthosessilioside F, white powder, C 36 H 54 O 11 . MS: [M] + 662.3751. 13 C NMR (100MHz, Pyridine-d5) δppm: 174.28, 173.15, 150.06, 147.68, 113.51, 111.08, 95.38, 79.52, 78.82, 75.42, 74.82, 74.21, 71.12, 70.52, 62.51, 62.12 ,49.65,47.55,44.33,44.31,44.12,42.23,41.65,41.36,38.85,35.21 ,33.45,32.47,28.91,26.72,25.24,23.15,19.27,18.92,17.79,13.83.

[0099] Elesesterpene G, white powder, C 29 H 42 O6. MS: [M] + 486.3058. 13 C NMR (100 MHz, Pyridine-d5) δppm: 211.13, 178.52, 172.81, 147.75, 113.86, 75.33, 70.58, 56.88, 51.98, 49.58, 49.31, 44.18, 44.05, 42.1 7. 41.77, 38.79, 37.12, 34.85, 34.41, 32.65, 32.59, 29.21, 29.15, 28.85, 25.37, 23.51, 18.75, 17.34, 13.71.

[0100] Acanthosessilioside K, white powder, C51 H 80 O 20 . MS: [M] + 1012.5521. 13 C NMR (100 MHz, Pyridine-d5) δppm: 174.52, 174.29, 151.25, 148.75, 113.68, 110.76, 105.02, 102.62, 95.38, 78.81, 78.21, 78.15, 77.18, 76.54, 76.51, 75.32, 74.19, 73.91, 72.73, 72.53, 71.59, 70.34, 69.51, 69.35, 64 .52, 63.18, 61.26, 50.41, 47.55, 44.31, 43.21, 41.92, 41.23, 40.18, 39.51, 38.32, 34.81, 34.71, 31.06, 29.97, 26.45, 25.92, 25.71, 23.67, 20.41, 19.42, 19.32, 18.47, 16.31, 14.92, 13.73.

[0101] Table 2 Abbreviations of drugs and compounds used

[0102]

[0103] Example 1 Lupane derivatives protect against ototoxicity induced by ototoxic drugs by maintaining cytoskeletal actin homeostasis

[0104] 1. Experimental Materials

[0105] (1) Lupane derivatives

[0106] Lupine derivatives are Chiisanoside, Chiisanogenin, Divaroside, Sessiloside A1, Elesesterpene F, Elesesterpene G, Acanthosessilioside F, Acanthosessilioside K, 1-Deoxychiisanoside, 24-Hydroxychiisanoside, 22α-Hydroxychiisanoside and 22α-Hydroxychiisanogenin.

[0107] (2) Main experimental reagents

[0108] Cisplatin (Sigma-Aldrich, Germany), gentamicin (Beijing Pulilai Gene Technology Co., Ltd.), chloramphenicol (Aladdin reagent), sodium salicylate (Sinopharm Chemical Reagent Beijing Co., Ltd.), insulin (Sigma-Aldrich, Germany), furosemide (Jiangsu Yabang Epson Pharmaceuticals), aconitine (Efa Biotechnology), DMEM culture medium (Hai Klon Biotechnology); fetal bovine serum (Clark, Australia); double antibody, trypsin, trypsin (EDTA-free); phosphate-buffered saline (PBS); CCK-8 (GLPBIO, USA); ready-to-use PI staining solution (Beijing Coolbo); Annexin V-FITC / PI double staining cell apoptosis detection kit (Guangzhou Beibo); 1% crystal violet staining solution, DAPI solution, FDA (Beijing Solebao Biotechnology Co., Ltd.), Trizol reagent, RIPA lysis buffer (Beijing Solebao Biotechnology Co., Ltd.), ECL chemiluminescence kit, Dock1 inhibitor TBOPP (Sigma-Aldrich, Germany).

[0109] 2. Experimental methods

[0110] 2.1 Experimental cell lines

[0111] Cochlear hair cells (HEI-OC1) were donated by the Third Hospital of Jilin University.

[0112] 2.2 Effects of lupane derivatives on HEI-OC1 cell viability

[0113] The cells were cultured using conventional methods, and the CCK-8 method was used to detect the effects of different concentrations of lupane derivatives (6.25, 12.5, 25, 50, and 100 µM) on the viability of HEI-OC1 cells.

[0114] 2.3 Effects of lupane derivatives on cell activity after ototoxic drug-induced cochlear hair cell damage

[0115] 2.3.1 Effects of lupane derivatives on cell activity after anti-tumor drug-induced cochlear hair cell damage

[0116] The cells were cultured using conventional methods, and were pre-treated with different concentrations of lupine derivatives (0, 6.25, 12.5, 25, 50, and 100 µM). After 24 h, cisplatin (50 µM, determined by experimental exploration) was used to induce cell damage. After 12 h, the CCK-8 method was used to determine the viability of HEI-OC1 cells.

[0117] 2.3.2 Effects of lupane derivatives on cell activity after aminoglycoside-induced cochlear hair cell damage

[0118] The cells were cultured using conventional methods, and were pre-treated with different concentrations of lupine derivatives (0, 6.25, 12.5, 25, 50, and 100 µM). After 24 h, gentamicin (500 µM, determined by experimental exploration) was used to induce cell damage. After 12 h, the CCK-8 method was used to determine the viability of HEI-OC1 cells.

[0119] 2.3.3 Effects of lupane derivatives on cell viability after non-aminoglycoside antibiotic-induced cochlear hair cell damage

[0120] The cells were cultured using conventional methods, and were pre-treated with different concentrations of lupine derivatives (0, 6.25, 12.5, 25, 50, and 100 µM). After 24 h, chloramphenicol (0.3 µM, determined by experimental exploration) was used to induce cell damage. After 12 h, the CCK-8 method was used to determine the viability of HEI-OC1 cells.

[0121] 2.3.4 Effects of lupane derivatives on cell viability after salicylate-induced cochlear hair cell damage

[0122] The cells were cultured using conventional methods, and were pre-treated with different concentrations of lupine derivatives (0, 6.25, 12.5, 25, 50, and 100 µM). After 24 h, sodium salicylate (20 µM, determined by experimental exploration) was used to induce cell damage. After 12 h, the CCK-8 method was used to determine the viability of HEI-OC1 cells.

[0123] 2.3.5 Effects of lupane derivatives on cell activity after diuretic-induced cochlear hair cell damage

[0124] The cells were cultured using conventional methods, and were pre-treated with different concentrations of lupine derivatives (0, 6.25, 12.5, 25, 50, and 100 µM). After 24 hours, furosemide (12.5 μM, determined by experimental exploration) was used to induce cell damage. After 12 hours, the CCK-8 method was used to determine the viability of HEI-OC1 cells.

[0125] 2.3.6 Effects of lupane derivatives on cell activity after insulin-induced cochlear hair cell damage

[0126] The cells were cultured using conventional methods, and were pre-treated with different concentrations of lupine derivatives (0, 6.25, 12.5, 25, 50, and 100 µM). After 24 h, insulin (1 μM, determined by experimental exploration) was used to induce cell damage. After 12 h, the CCK-8 method was used to determine the viability of HEI-OC1 cells.

[0127] 2.3.7 Effects of lupane derivatives on cell viability after aconitine-induced cochlear hair cell damage

[0128] The cells were cultured using conventional methods, and were pre-treated with different concentrations of lupine derivatives (0, 6.25, 12.5, 25, 50, and 100 µM). After 24 h, aconitine (1 μM, determined by experimental exploration) was used to induce cell damage. After 12 h, the viability of HEI-OC1 cells was determined using the CCK-8 method. 2.4 Crystal violet staining

[0129] 7×10³ HEI-OC1 cells were inoculated into 96-well plates and incubated at 37°C and 5% CO2 for 24h. After pretreatment with medium containing different concentrations (12.5, 25 and 50μM) of Chiisanoside for 2h, a cisplatin injury model was established. The cells were rinsed twice with PBS, fixed with fixative for 30 minutes, and air-dried. They were stained with 0.1% crystal violet for 20 minutes, the stain was removed, and the cells were rinsed twice with PBS. The number and morphology of cells were observed under a fluorescence microscope.

[0130] 2.5 Flow cytometric analysis of apoptosis

[0131] Cells were plated at 5 × 10 4 Cells were inoculated in 6-well plates at a density of 100 μM and cultured for about 24 h. The culture medium containing different concentrations of Chiisanoside (12.5, 25, and 50 μM) was replaced. After washing, the cells were suspended in Annexin V binding solution, and then incubated with Annexin V-FITC staining solution (protected from light, 15 min). Then, PI staining solution was added and continued to be incubated (protected from light, 5 min). The cells were filtered through a 200-mesh filter into a flow tube and immediately detected by flow cytometry (completed within one hour to avoid fluorescence quenching). The data were then analyzed using FlowJo10 software.

[0132] 2.6 Transcriptomics sequencing

[0133] HEI-OC1 cells were collected at 1×10 6 The cells were seeded in 6-well plates at a density of 1.5 μM / well for 12 h and then pretreated with the test drug (optimal concentration: 25 μM) for 2 h, while the control group was not given any drug. The cells were then subjected to the cisplatin model and the following operations were performed.

[0134] 2.6.1 RNA extraction and identification

[0135] After 2 hours of treatment with the test drug and establishment of the cisplatin model, total RNA was extracted from HEI-OC1 cells using TRIzol reagent. RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).

[0136] 2.6.2 qRT-PCR

[0137] Total RNA (Trizol extraction) was taken from each group, and after the concentration was determined, cDNA was synthesized according to the instructions of the reverse transcription kit, and the reaction system was prepared. cDNA amplification was performed using a qRT-PCR instrument, and the relative expression level of the target gene was calculated according to the Ct value.

[0138] 2.7 Cytoskeletal actin staining

[0139] HEI-OC1 cells were cultured at 5×10 4 / well density was seeded in 24-well plates and the cisplatin-induced injury model was established after pretreatment with Chiisanoside (12.5, 25 and 50 μM). Alexa Fluor 488-conjugated phalloidin, Alexa Fluor 594-conjugated DNase I and DAPI were used to visualize F-actin, G-actin and nucleus of HEI-OC1 cells, respectively.

[0140] 2.8 Cytoskeletal actin polymerization activity

[0141] Actin filament polymerization activity in each fraction cell was assessed using a commercially available kit (Actin Polymerization Kit #BK003, Cytoskeleton Inc.).

[0142] 2.9 Analysis of related protein expression

[0143] 2.9.1 Extraction of total cell protein

[0144] HEI-OC1 cells were inoculated in 6-well plates and pretreated with 25µM of representative compound Chiisanoside for 24h. The culture medium was discarded, and the cells were washed three times with pre-cooled PBS. 200μL of cell lysis buffer was added. After the cells were completely lysed, they were scraped and aspirated into a 1.5mL centrifuge tube. Centrifuged at 4℃ and 12500rpm for 25min, the supernatant was collected after the precipitate was removed, and stored at -80℃ (cell lysis was performed on an ice box).

[0145] 2.9.2 BCA method for protein concentration determination

[0146] The protein concentration of each sample was detected using a BCA kit.

[0147] 2.9.3 Kit detection of related protein expression

[0148] The protein expression of representative compound Chiisanoside (12.5, 25 and 50 μM) and DOCK1 inhibitor group (TBOPP+Chiisanoside 25μM, abbreviated as TBOPP+CSS-M) was detected using Dock1, PIP5K1A and Gelsolin protein content detection kits. Protein quantification was performed by BCA method, followed by SDS-PAGE gel electrophoresis, membrane transfer and blocking. The corresponding primary and secondary antibodies were added in sequence, and the luminescent solution was added before development on a developer. β-actin was used as a control, and the protein blot was analyzed using Image J software.

[0149] 2.10 Statistical analysis

[0150] The data were analyzed using Prism 9 statistical software and expressed as Mean ± SD. The groups were compared using t test combined with one-way analysis of variance. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001.

[0151] 3. Experimental results

[0152] 3.1 Effects of lupane derivatives on the viability of cochlear hair cells

[0153] Through measurement, the lupine derivatives of the present invention (0, 6.25, 12.5, 25, 50, 100 µM) did not show obvious cytotoxic activity against HEI-OC1 cells, but showed certain proliferative activity. The results are shown in Figure 1 .

[0154] 3.2 Protective activity of lupane derivatives on cochlear hair cells

[0155] From the perspective of cell protection activity against HEI-OC1, the lupine derivatives all showed good protective effects against ototoxic drug-induced cytotoxicity, among which Chiisanoside, Divaroside, Sessiloside A1 and Chiisanogenin had the best activity. After administration, the cell activity was even twice as high as that of the model group. The cell activity results are shown in Figure 2 (Taking cisplatin model as an example), its EC 50 See Tables 3-9 for values.

[0156] Table 3 EC protection of HEI-OC1 cells in cisplatin model 50 Value (μM)

[0157]

[0158] Table 4 EC protection of HEI-OC1 cells in gentamicin model50 Value (μM)

[0159]

[0160] Table 5 EC protection of HEI-OC1 cells under chloramphenicol model 50 Value (μM)

[0161]

[0162] Table 6 EC of HEI-OC1 cells protected by sodium salicylate model 50 Value (μM)

[0163]

[0164] Table 7 EC of HEI-OC1 cells in the furosemide protection model 50 Value (μM)

[0165]

[0166] Table 8 EC of HEI-OC1 cells under insulin protection model 50 Value (μM)

[0167]

[0168] Table 9 EC of HEI-OC1 cells under the protection of aconitine model 50 Value (μM)

[0169]

[0170] In the following, only Chiisanoside (25 μM) is used as a representative, and only the operation and results of the representative modeling drug cisplatin are given.

[0171] 3.3 Effect of Chiisanoside on HEI-OC1 Cell Morphology

[0172] 3.3.1 Crystal violet staining

[0173] The crystal violet staining results of HEI-OC1 cells in each group are shown in Figure 3. The number of living cells in the model group was less than that in the control group, and the cytoskeleton was damaged and deformed; the cell viability in the Chiisanoside pretreatment group was improved in a dose-dependent manner, reaching a peak at 25μM, and the cytoskeleton damage was reversed, with neat arrangement and regular morphology.

[0174] 3.3.2 Effect of Chiisanoside on apoptosis of HEI-OC1 cells

[0175] Flow cytometric analysis Figure 4As shown in the figure, the percentage of apoptosis and necrosis of HEI-OC1 cells after cisplatin treatment increased significantly to 50% (p < 0.005), compared with the model group, while the apoptosis and necrosis rates were reduced to 40.09% (p < 0.05), 25.84% (p < 0.005) and 11.38% (p < 0.005) after pretreatment with 12.5, 25 and 50 μM Chiisanoside, respectively. These results indicate that Chiisanoside can effectively improve cisplatin-induced apoptosis and necrosis of HEI-OC1 cells and play a pre-protective role against cisplatin-induced ototoxicity.

[0176] 3.4 Transcriptome differential analysis

[0177] DEGs in the model group (M) and chiisanoside group (CSS) were enriched by KEGG pathways. Figure 5 As shown, the top 20 pathways with the most reliable enrichment significance (i.e., the smallest Q value) are selected to present the results. Among them, the present invention selects the most relevant and most significantly different PI3K / AKT, actin cytoskeleton regulation and apoptosis signaling pathways. Through the volcano plot, it was found that Chiisanoside can significantly counteract the changes in the expression levels of the above-mentioned pathway-related genes Dock1, PIP5K1A, Gelsolin, GPX4, SLC7A11, and TGFBR2 induced by cisplatin. The results are shown in Figure 6 , Figure 7 shown.

[0178] 3.5 qRT-PCR

[0179] The qRT-PCR primer sequences are shown in Table 10

[0180] Table 10 Primer sequence information

[0181]

[0182] 3.6 Cytoskeletal actin staining

[0183] To explore the protective effect of Chiisanoside on cisplatin-induced cytoskeletal damage, F-actin (green), G-actin (red) and Dapi (blue) of cisplatin-induced HEI-OC1 cells were simultaneously stained and observed. Figure 8 As shown, after cisplatin induction, the cytoskeleton was disordered, significantly damaged, irregular in shape, and the amount of depolymerized G-actin increased; the cells in the Chiisanoside pretreatment group had regular shapes, less cytoskeleton damage, and significantly less depolymerized G-actin than the model group; the addition of the Dock1 inhibitor group reversed the pre-protection phenomenon of Chiisanoside.

[0184] 3.7 Cytoskeletal actin polymerization activity

[0185] Since GSN activation cuts off F-actin, the cytoskeleton homeostasis is disturbed. The activity of HEI-OC1 cytoskeleton actin polymerization before and after cisplatin treatment is shown in Figure 9. The activity of the cisplatin treatment group is significantly reduced, and the activity of the Chiisanoside pretreatment group is significantly increased in a dose-dependent manner, reaching a peak at 25μM. After adding the Dock1 inhibitor, the activity is significantly reduced again.

[0186] 3.8 Determination of related protein expression

[0187] The results of western blotting for the activity verification of related proteins are as follows Fig.10 As shown in the results, the protein expression of HEI-OC1 increased after ototoxic drugs induced damage, while the expression of Dock1 and PIP5K1A decreased. Chiisanoside can increase the expression of Dock1 and PIP5K1A and inhibit the expression of Gelsolin protein. The experiment showed that lupine derivatives anti-ototoxic drugs induced apoptosis of cochlear hair cells and regulated the actin cytoskeleton, and Chiisanoside was the most significant, which upregulated the expression of Dock1 and PIP5K1A, reduced the content of PI4P, increased the content of PIP2 and inhibited the expression of Gelsolin protein. Dock1 inhibitor reversed the protective activity, indicating that Chiisanoside protected HEI-OC1 and mouse cochlear hair cells from irreversible damage caused by cisplatin-induced actin depolymerization through the Dock1 / PIP5K1A / Gelsolin pathway.

[0188] Example 2 Chiisanoside inhibits ototoxic drug-induced ferroptosis of otic hair cells by activating the TGFBR2 / SLC7A11 / GPX4 pathway

[0189] 1. Experimental Materials

[0190] (1) Lupane derivatives

[0191] Same as "(1) Lupane derivatives" in Example 1.

[0192] (2) Main experimental reagents

[0193] Cell-related reagents were the same as those in “(2) Main experimental reagents” in Example 1; DAPI solution, JC-1, FDA, LDH, HMBG-1, GSH, 4-HNE, MDA, PI4P, PIP2 (Beijing Solebold Biotechnology Co., Ltd.), Fe 2+Kit (Shanghai Andy Biotechnology Co., Ltd.), Trizol reagent, RIPA lysis buffer (Beijing Solebow Biotechnology Co., Ltd.), ECL chemiluminescence kit, Ros kit (Shanghai Bio Yuntian Biotechnology Co., Ltd.).

[0194] 2. Experimental methods

[0195] 2.1 Experimental cell lines

[0196] Same as “2.2 Experimental cell lines” in Example 1.

[0197] 2.2 ROS content analysis

[0198] HEI-OC1 cells were cultured at 5×10 4 The wells were seeded at a density of 1.5 μM in 24-well plates, and the cisplatin-induced injury model was established after pretreatment with the representative compound Chiisanoside (12.5, 25, and 50 μM).

[0199] 2.3 Detection of relevant biochemical indicators

[0200] Measurement of LDH, HMBG-1, PI4P, PIP2, MDA, 4-HNE, GSH, Fe 2+ HEI-OC1 cells (15×10 4 Cells were then collected and the culture medium was used to detect LDH and HMBG-1, and cells were lysed and used for PI4P, PIP2, MDA, 4-HNE, GSH, and Fe 2+ of detection kits.

[0201] 2.4 Protein expression analysis

[0202] 2.4.1 Extraction of total cell protein

[0203] The operation is the same as that in “2.11.1 Extraction of total cell protein” in the example.

[0204] 2.4.2 Determination of protein concentration by BCA method

[0205] The protein concentration of each sample was detected using a BCA kit.

[0206] 2.4.3 Kit detection of related protein expression

[0207] The protein expression of representative compound Chiisanoside (12.5, 25 and 50 μM) and overexpression group (shTGFBR2+Chiisanoside 25 μM, abbreviated as shTGFBR2+CSS-M) was detected using TGFBR2, SLC7A11 and GPX4 protein content detection kits. Protein quantification was performed by BCA method, followed by SDS-PAGE gel electrophoresis, membrane transfer, and blocking. The corresponding protein primary and secondary antibodies were added in sequence, and the luminescent solution was added and developed on a developer. β-actin was used as a control, and the protein blot was analyzed using Image J software.

[0208] 2.5 Statistical analysis

[0209] All data were analyzed using Prism 9 (GraphPad) statistical software. The data were expressed as Mean ± SD. The groups were compared using t-test combined with one-way analysis of variance. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001.

[0210] 3. Experimental results

[0211] 3.1 ROS content analysis

[0212] The results of ROS content analysis are as follows Fig.11 As shown, the fluorescence (green) intensity in the cisplatin-treated group was significantly enhanced compared with the control group (p < 0.001), indicating the overproduction of ROS in HEI-OC1 cells. However, when the cells were pretreated with Chiisanoside, the fluorescence intensity decreased in a dose-dependent manner, and the effect was very significant at 25 μM.

[0213] 3.2 Biochemical index detection

[0214] The results of the relevant impact factor level test are as follows Fig.12 , Fig.13 The invention measures the changes of ferroptosis biomarkers, and cisplatin induces MDA, 4-HNE and Fe in HEI-OC1 cells. 2+ The level of Fe in the Chiisanoside pretreatment group was increased, and the level of GSH was decreased. 2+, MDA, and 4-HNE accumulation decreased, and GSH levels increased in a dose-dependent manner. The results showed that Chiisanoside pretreatment could reverse the changes in ferroptosis markers of HEI-OC1 cells induced by cisplatin. At the same time, the changes in cytoskeleton actin-related markers were also detected. Cisplatin induced an increase in PI4P, HMBG-1, and LDH, and a decrease in PIP2 in HEI-OC1 cells. Chiisanoside treatment reversed the above phenomenon, and TBOPP, a selective inhibitor of Dock1, blocked the above effects of chiisanoside.

[0215] 3.3 Determination of related protein expression

[0216] The results of related protein expression determination were as follows Fig.14 As shown. After HEI-OC1 cells were induced by ototoxic drugs, the expression of TGFBR2 increased significantly, while the expression of SLC7A11 and GPX4 decreased significantly, which was the main cause of intracellular lipid peroxide accumulation and ferroptosis. Compared with the model group, the expression of TGFBR2 in the Chiisanoside pretreatment group decreased significantly with the increase of dose, reaching a peak at 25μM, and the expression of SLC7A11 and GPX4 gradually increased, showing a dose-dependent trend.

[0217] The above results indicate that Chiisanoside inhibits the occurrence of cisplatin-induced ferroptosis in HEI-OC1 cells by inhibiting the expression of TGFBR2 protein, thereby activating SLC7A11 / GPX4 and decomposing accumulated lipid peroxides.

[0218] Example 3 Effect of Chiisanoside on Mice with Hearing Loss Induced by Ototoxic Drugs

[0219] 1. Experimental Materials

[0220] Cisplatin (Sigma-Aldrich, Germany); mouse cochlear hair cells HEI-OC1 (provided by the Third Hospital of Jilin University); 1,2-propanediol (Beijing Chemical Plant); sodium carboxymethyl cellulose (Shanghai Aladdin Biochemical Technology Co., Ltd.); Dock1, PIP5K1A, Gelsolin, TGFBR2, SLC7A11 and GPX4, β-actin antibodies (Abcam, UK); Dock1 inhibitor TBOPP (Sigma); RIPA lysis buffer (Beijing Solebold Technology Co., Ltd.); BCA kit (Shanghai Bio-Technology Co., Ltd.); TRIzol reagent (Beijing Solebold Technology Co., Ltd.); qRT-PCR kit (Takara, Japan).

[0221] 2. Experimental methods

[0222] 2.1 Experimental animals

[0223] Adult male C57BL / 6 mice, weighing 20-22 g, were obtained from Changchun Yisi Experimental Animal Technology Co., Ltd. and raised in a controlled laboratory environment. They were randomly divided into groups, with 8 mice in each group.

[0224] 2.2 Establishment of cisplatin ototoxicity mouse model

[0225] Adult male C57BL / 6 mice were selected and divided into five groups of equal number, namely control group, cisplatin group (CDPP), high-dose treatment group (CSS-H), medium-dose treatment group (CSS-M), and low-dose treatment group (CSS-L). The control group was intraperitoneally injected with 6 ml / kg of normal saline for 5 consecutive days; the experimental group was intraperitoneally injected with 6 mg / kg of cisplatin for 5 consecutive days; the high-dose group, medium-dose group, and low-dose group were intragastrically administered with Chiisanoside monomer compound 200 mg / kg, 100 mg / kg, and 50 mg / kg, respectively, for 7 consecutive days; from the 8th day, Chiisanoside was intragastrically administered 2 hours later and then 6 mg / kg of cisplatin was intraperitoneally injected for 5 consecutive days.

[0226] 2.3 Auditory brainstem evoked potential (ABR) detection of mouse hearing threshold

[0227] After administration, ABR tests were performed on the mice to observe changes in their hearing.

[0228] 2.4 Determination of related protein expression

[0229] The representative drug Chiisanoside was used at a dose of 200 mg / kg to pre-treat the expression levels of Dock1, PIP5K1A, Gelsolin, TGFBR2, SLC7A11, GPX4 and other proteins in the mouse tissue homogenate. The protein was quantified by the BCA method, followed by SDS-PAGE gel electrophoresis, membrane transfer, and blocking. The corresponding primary and secondary antibodies were added in sequence, and the luminescent solution was added before development on a developer. β-actin was used as a control, and protein blots were analyzed using Image J software.

[0230] 2.5 Statistical analysis

[0231] SPSS statistical software package was used for t-test and analysis of variance.

[0232] 3. Results

[0233] 3.1 Hearing test of mice with cisplatin-induced ototoxicity

[0234] The hearing test results of the cisplatin model group were monitored after intraperitoneal injection of cisplatin. Fig.15As shown in the figure, the incidence of hearing loss was 100%, indicating that the deaf mouse model was successfully established. Compared with the model group, the lupine derivatives (200 mg / kg) in each group could significantly reduce the ototoxicity caused by cisplatin. It is worth noting that the chiisanoside group could significantly inhibit the ototoxicity caused by cisplatin.

[0235] 3.2 Activity verification of expression levels of cytoskeletal actin-related proteins

[0236] The results of the expression levels of cytoskeletal actin-related proteins are shown in Figure 16 , Fig.17 As shown, cisplatin induced a decrease in the expression of Dock1 and PIP5K1A in the cochlea of ​​mice, and an increase in PI4P and a decrease in PIP2, which led to an increase in the expression of Gelsolin protein. In the Chiisanoside pretreatment group, the expression of Dock1 and PIP5K1A increased significantly with the increase in dose, reaching a peak at 25μM, which reduced PI4P and increased PIP2, inhibiting its activation by binding to Gelsolin protein and maintaining the homeostasis of cytoskeletal actin. Dock1 inhibitor reversed the effect of Chiisanoside on the Dock1 / PIP5K1A / Gelsolin signaling pathway, which was consistent with the results of cell experiments, indicating that Chiisanoside activated Dock1 / PIP5K1A to convert PI4P into PIP2, inhibiting the activation of Gelsolin protein, and inhibiting the irreversible damage caused by cisplatin-induced cytoskeletal actin depolymerization.

[0237] 3.3 Expression levels of ferroptosis-related proteins in mouse cochlea

[0238] To determine the protective effect of chiisanoside on cisplatin-induced cell ferroptosis through the TGFBR2 / SLC7A11 / GPX4 signaling pathway, Western blotting was used to evaluate the expression levels of related proteins. Fig.17 , Fig.18 As shown in the figure, cisplatin-induced TGFBR2 expression in the cochlea of ​​mice was significantly increased, and SLC7A11 and GPX4 expression was significantly decreased, which was the main cause of ferroptosis. In the chiisanoside pretreatment group, TGFBR2 expression was decreased, and SLC7A11 and GPX4 expression was increased in a dose-dependent manner. This indicates that chiisanoside may inhibit cisplatin-induced cochlear hair cell ferroptosis by inhibiting TGFBR2 protein expression and activating SLC7A11 / GPX4.

[0239] The above experimental results indicate that Chiisanoside protects HEI-OC1 and mouse cochlear hair cells from irreversible damage caused by cisplatin-induced actin depolymerization through the Dock1 / PIP5K1A / Gelsolin pathway, and effectively activates the TGFBR2 / SLC7A11 / GPX4 signaling pathway to resist cisplatin-induced cell ferroptosis, thereby playing a pre-protective role against cisplatin-induced ototoxicity.

[0240] Example 4 Preparation of Chiisanoside Tablets

[0241] Reagents: starch (pharmaceutical grade, Tianjin Jindong Tianzheng Fine Chemical Reagent Factory); citric acid (Shanghai McLean Biochemical Technology Co., Ltd.); magnesium stearate (Shanghai McLean Biochemical Technology Co., Ltd.).

[0242] Preparation method:

[0243] ① Preparation of 10% starch slurry: Dissolve 0.25 g citric acid in 25 mL pure water, add 2.5 g starch and disperse evenly, heat to gelatinize, and obtain 10% starch slurry.

[0244] ② Granulation: Mix 20 g of Chiisanoside powder and 20 g of starch evenly, then add 10% starch slurry and mix, grind evenly, make a soft material, sieve through a 16-mesh sieve for granulation, and dry at 60°C for 1 h. After granulation through a 16-mesh sieve, add lubricant magnesium stearate and press into tablets with a shallow punch with a diameter of 10 mm.

[0245] The obtained tablets are off-white in color, uniform in color, uniform in thickness, and moderate in hardness. The tablet weight and disintegration time meet the requirements. The obtained Chiisanoside tablets meet the requirements and can be used as tablets.

[0246] Example 5 Preparation of Chiisanoside Suspension Injection

[0247] Reagents: polylactic acid (PLA, Shanghai Zhenzhun Biotechnology Co., Ltd.); polylactic acid-co-glycolic acid (PLGA, Shanghai Yuanye Biotechnology Co., Ltd.); poloxamer 188 (Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.); dichloromethane, methanol, acetonitrile, etc. (Tianjin Tiantai Chemicals Co., Ltd.).

[0248] Preparation method:

[0249] Preparation of polymer microparticles: Weigh 2.5 g of Chiisanoside and 2.5 g of carrier (PLA / PLGA) into a 50 mL round-bottom flask, add 5 mL of dichloromethane to dissolve, remove most of the organic solvent by reduced pressure distillation at 28°C, and then vacuum dry at 40°C for 24 h until the solvent is completely removed, grind, and pass through a sieve with a pore size of 150 μm to obtain Divaroside polymer microparticles.

[0250] Preparation of Chiisanoside suspension injection: 2.5 g Chiisanoside polymer microparticles were dispersed in 250 mL of aqueous solution containing 10 g / L poloxamer 188 stabilizer under continuous stirring until the dispersion was complete. The drug dispersion was ground to the desired particle size, and the Chiisanoside polymer microparticle suspension was obtained and stirred at 3000 r·min -1 The mixture was centrifuged for 1 min and dispersed with 10 mL of aqueous stabilizer solution to concentrate the preparation to about 25 g / L.

[0251] The obtained suspension injection has uniform particle size, and the water content and surface particle size of the preparation meet the requirements. The in vitro sustained release effect is good and the stability is good. The obtained Chiisanoside suspension injection meets the requirements and can be used as a suspension injection.

[0252] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of lupine derivatives in the preparation of drugs for preventing and / or treating drug-induced deafness, characterized in that: The lupine derivative has a general structure as shown in Formula I: wherein R1 is selected from -H, -β-D-glucose, -β-D-glucose-(6←1)-β-D-glucose or -β-D-glucose-(6←1)-β-D-glucose-(6←1)-α-L-rhamnose; R2 is selected from -H, -OH or -O(CH2)2CH3; R3 is selected from -CH3 or -CH2OH; R4 is selected from -OH or -H; R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

2. Use of lupine derivatives in the preparation of drugs for inhibiting the instability of hair cell skeleton actin induced by ototoxic drugs, characterized in that: The lupine derivative has a general structure as shown in Formula I: wherein R1 is selected from -H, -β-D-glucose, -β-D-glucose-(6←1)-β-D-glucose or -β-D-glucose-(6←1)-β-D-glucose-(6←1)-α-L-rhamnose; R2 is selected from -H, -OH or -O(CH2)2CH3; R3 is selected from -CH3 or -CH2OH; R4 is selected from -OH or -H; R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

3. Use of lupine derivatives in the preparation of drugs for inhibiting hair cell ferroptosis induced by ototoxic drugs, characterized in that: The lupine derivative has a general structure as shown in Formula I: wherein R1 is selected from -H, -β-D-glucose, -β-D-glucose-(6←1)-β-D-glucose or -β-D-glucose-(6←1)-β-D-glucose-(6←1)-α-L-rhamnose; R2 is selected from -H, -OH or -O(CH2)2CH3; R3 is selected from -CH3 or -CH2OH; R4 is selected from -OH or -H; R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

4. The use according to any one of claims 1 to 3, characterized in that: The lupetane derivatives include Chiisanoside, Chiisanogenin, Divaroside, Sessiloside A1, Elesterpene F, Elesterpene G, Acanthosessilioside F, Acanthosessilioside K, 1-Deoxychiisanoside, 24-Hydroxychiisanoside, 22α-Hydroxychiisanoside and 22α-Hydroxychiisanogenin.

5. The use according to claim 4, characterized in that The drug-induced deafness includes cisplatin-induced deafness.

6. A pharmaceutical composition, characterized in that A lupine derivative comprising a chemical structure shown in the following formula I, and a pharmaceutically acceptable excipient; wherein R1 is selected from -H, -β-D-glucose, -β-D-glucose-(6←1)-β-D-glucose or -β-D-glucose-(6←1)-β-D-glucose-(6←1)-α-L-rhamnose; R2 is selected from -H, -OH or -O(CH2)2CH3; R3 is selected from -CH3 or -CH2OH; R4 is selected from -OH or -H; R5 is selected from -CH(CH2)CH3, -C(O)CH3 or -CH(CH3)COOH.

7. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the pharmaceutical composition includes pills, suspension injections, tablets, granules, pastes, capsules, gels, drops or inhalants.

8. The pharmaceutical composition according to claim 6, characterized in that The lupetane derivatives include Chiisanoside, Chiisanogenin, Divaroside, Sessiloside A1, Elesterpene F, Elesterpene G, Acanthosessilioside F, Acanthosessilioside K, 1-Deoxychiisanoside, 24-Hydroxychiisanoside, 22α-Hydroxychiisanoside and 22α-Hydroxychiisanogenin.

9. The pharmaceutical composition according to claim 8, characterized in that The lupane derivative is chiisanoside.

10. Use of the pharmaceutical composition according to any one of claims 6 to 9 in the preparation of a drug for preventing and / or treating drug-induced deafness, characterized in that: The drug-induced deafness includes cisplatin-induced deafness.

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