Use of a triterpenoid saponin and its analogues in the preparation of a medicament for treating presbycusis

Abstract

The present invention discloses the application of a triterpenoid saponin and its analogues in the preparation of a medicament for treating presbycusis, belonging to the technical field of biomedicine. The triterpenoid saponin and its analogues include Chiisanoside, Divaroside, Sessiloside A1 or Chiisanogenin. The triterpenoid saponin and its analogues provided by the present invention are derived from the leaves and fruits of Eleutherococcus senticosus and Eleutherococcus sessiliflorus, which are rich in resources and relatively renewable, are rich in the original plants or can be obtained through simple transformation, are safe to operate, and have low production costs. The triterpenoid saponin and its analogues provided by the present invention achieve the effect of treating presbycusis by inhibiting the senescence of hair cells in the ear, can act precisely on the targets related to presbycusis, make the drug effect more direct and effective, and can effectively reduce the generation of toxic and side effects, providing reference data and new research and development ideas for the treatment of sensorineural presbycusis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of a triterpenoid saponin and its analogues in the preparation of a medicament for treating presbycusis. Background Art

[0002] Age-related hearing loss (ARHL), also known as presbycusis, is one of the most common sensory dysfunctional diseases in the elderly. ARHL is characterized by the initial damage to the high-frequency region of the auditory spectrum, which spreads to the low-frequency region as age increases. ARHL is a progressive, irreversible, and bilaterally symmetric hearing impairment, and its main cause may be due to the apoptosis of cochlear hair cells and cell senescence leading to cell damage. The occurrence of ARHL seriously affects the language communication ability of the elderly, reduces the quality of life, and is the main cause of social isolation and cognitive decline in the elderly. With the development of modern social aging, the incidence of presbycusis has also increased year by year. Some studies have shown that among people over 65 years old, about one-half of them have hearing loss to varying degrees. Currently, presbycusis mainly has the following several types: sensorineural, metabolic (stria vascularis), central, mixed, cochlear conductive, and undetermined types. Among them, the most common and main type is sensorineural presbycusis, which is mainly characterized by the damage of cochlear hair cells and manifested as age-related hearing loss with high-frequency hearing loss.

[0003] At present, the clinical treatment strategies for sensorineural presbycusis mainly include hearing aids, cochlear implants, drug therapy, auditory rehabilitation training, etc. A hearing aid is a device that improves hearing by amplifying sound. It can collect, amplify external sounds and transmit them into the ear, enabling patients to hear clearer sounds. However, it also amplifies surrounding noises, resulting in insignificant improvement in the hearing of patients in noisy environments. Long-term wearing can cause ear discomfort, such as earache, itching, etc. A cochlear implant is a method of restoring hearing through an electronic device implanted in the body. It converts external sounds into electrical signals and directly stimulates the auditory nerve, enabling patients to hear sounds. However, this surgery has certain risks, such as infection, bleeding, etc. Moreover, it is expensive and requires certain rehabilitation training after implantation. The effect is not ideal for some patients, such as those with severely degenerated auditory nerves. Some drugs may delay or improve sensorineural presbycusis by improving inner ear blood circulation, protecting auditory nerve cells, inhibiting inflammatory reactions, etc. For example, vasodilator drugs can increase blood supply to the inner ear, and neurotrophic drugs can promote the growth and repair of auditory nerve cells. However, there is currently no specific drug that can completely cure sensorineural presbycusis. The effect of drug therapy varies from person to person, and it needs to be taken for a long time, which may have side effects. In short, although the clinical treatment strategies for sensorineural presbycusis have certain effects, there are still many limitations. Therefore, it is necessary to further study its etiology and pathogenesis in depth and develop more effective treatment methods. Summary of the Invention

[0004] The object of the present invention is to provide an application of triterpenoid saponins and their analogs in the preparation of drugs for treating presbycusis, so as to solve the problems existing in the above-mentioned prior art. The triterpenoid saponins and their analogs extracted from Acanthopanax senticosus or Acanthopanax sessiliflorus can accurately act on the targets related to presbycusis, making the drug effect more direct and effective, and can effectively reduce the generation of toxic and side effects, providing reference data and new research and development ideas for the treatment of sensorineural presbycusis.

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

[0006] The present invention provides an application of triterpenoid saponins and their analogs in the preparation of drugs for treating presbycusis, and the triterpenoid saponins and their analogs have a general formula structure shown in Formula I:

[0007]

[0008] Among them, 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;

[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] Furthermore, the triterpenoid saponins and their analogs include Chiisanoside, Divaroside, Sessiloside A1 or Chiisanogenin.

[0014] Furthermore, the triterpenoid saponins and their analogs are Chiisanoside.

[0015] Furthermore, the dosage forms of the drug include oral preparations and injections.

[0016] Furthermore, the oral preparations include tablets.

[0017] Furthermore, the injections include suspension injections.

[0018] Furthermore, the presbycusis is sensorineural presbycusis.

[0019] Furthermore, the triterpenoid saponins and their analogs achieve the treatment of presbycusis by inhibiting the senescence of hair cells.

[0020] Furthermore, the triterpenoid saponins and their analogs inhibit the apoptosis and cell cycle arrest of hair cells, thereby inhibiting the senescence of hair cells.

[0021] The present invention also provides the use of the triterpenoid saponins and their analogs in the preparation of inhibitors of TRAF2, PERK, Caspase-3, DAXX, p-MKK3 or p-p38 MAPK.

[0022] The present invention also provides the use of the triterpenoid saponins and their analogs in the preparation of activators of CyclinE or CyclinE-CDK2.

[0023] The present invention discloses the following technical effects:

[0024] The triterpenoid saponins and their analogs provided by the present invention are all natural products, derived from the leaves and fruits of Eleutherococcus senticosus and Acanthopanax sessiliflorus which are rich in resources and relatively renewable. The natural products involved in the present invention are rich in the original plants or can be obtained through simple transformation, with safe operation and low production cost.

[0025] Through pharmacological experiments, it is proved that the triterpenoid saponins and their analogs extracted from Acanthopanax senticosus or Acanthopanax sessiliflorus inhibit the expression of TRAF2 / PERK / Caspase-3 proteins through precise multi-target actions, thereby preventing the apoptosis of hair cells in the ear caused by endoplasmic reticulum stress, Ca 2+ overload, mitochondrial dysfunction and damage, and a sharp increase in ROS content. At the same time, by inhibiting the expression of DAXX / MKK3 / p38 proteins and activating the expression of CyclinE protein, the kinase activity of CyclinE-CDK2 is activated, preventing hair cells in the ear from stagnating in the G1 phase and not entering the S phase, thereby preventing the characteristic changes of hair cell senescence. Its mechanism of action is clear, it can act precisely on the target, making the drug effect more direct and effective, and can effectively reduce the generation of toxic and side effects. Compared with the existing clinical treatment strategies, the triterpenoid saponins and their analogs provided by the present invention have both precise target correspondence and also reflect the multi-target overall intervention effect on diseases. The present invention provides reference data and new research and development ideas for the treatment of sensorineural presbycusis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 It is a statistical chart for detecting the effect of CSS and its analogs on the viability of HEI-OC1 cells;

[0028] Figure 2 It is a statistical chart for detecting the effect of CSS and its analogs on the viability of D-Gal-induced senescent HEI-OC1 cells;

[0029] Figure 3 It is the detection result of crystal violet staining of D-Gal-induced HEI-OC1 cells by CSS;

[0030] Figure 4 It is the detection result of FDA / PI staining of D-Gal-induced HEI-OC1 cells by CSS;

[0031] Figure 5 It is the detection result of flow cytometry of D-Gal-induced HEI-OC1 cells by CSS;

[0032] Figure 6 It is a KEGG analysis diagram of differential genes in transcriptome sequencing of D-Gal-induced HEI-OC1 cells by CSS;

[0033] Figure 7 Statistical chart of the effects of CSS on the expression of apoptosis- and senescence-related genes TRAF2 (A), PERK (B), Caspase-3 (C), DAXX (D), MKK3 (E), p38 MARK (F), and CyclinE (G) in D-Gal-induced HEI-OC1 cells;

[0034] Figure 8 Statistical chart of the detection results of the effects of CSS on ROS in D-Gal-induced HEI-OC1 cells;

[0035] Figure 9 Statistical chart of the detection results of the effects of CSS on the mitochondrial membrane potential of D-Gal-induced HEI-OC1 cells;

[0036] Figure 10 For the related biochemical indexes Ca of CSS on D-Gal-induced HEI-OC1 cells 2+ (A), MDA (B), SOD (C), LDH (D), HMBG-1 (E), and GSH (F) statistical chart of the detection results;

[0037] Figure 11 Statistical chart of the detection results of the expression of apoptosis-related proteins TRAF2 (A), PERK (B), and Caspase-3 (C) in CSS-treated D-Gal-induced HEI-OC1 cells;

[0038] Figure 12 Statistical chart of the detection results of the cell cycle of CSS-treated D-Gal-induced HEI-OC1 cells;

[0039] Figure 13 Statistical chart of the SA-β-gal staining results of CSS-treated D-Gal-induced HEI-OC1 cells;

[0040] Figure 14 Statistical chart of the detection results of the expression of senescence-related proteins DAXX (A), MKK3 (B), p38 MARK (C), and CyclinE (D) in CSS-treated D-Gal-induced HEI-OC1 cells;

[0041] Figure 15 Statistical chart of the ABR detection results of CSS-treated D-Gal-induced senescent mice;

[0042] Figure 16 Statistical chart of the basement membrane spreading staining results of CSS-treated D-Gal-induced senescent mice;

[0043] Figure 17It is a statistical chart of the detection results of the expressions of apoptosis-related proteins TRAF2 (A), PERK (B), and Caspase-3 (C) in CSS-treated D-Gal-induced senescent mice;

[0044] Figure 18 It is a statistical chart of the detection results of the expressions of cell cycle regulation-related proteins DAXX (A), MKK3 (B), p38MARK (C), and CyclinE (D) in CSS-treated D-Gal-induced senescent mice. Detailed implementation manners

[0045] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0049] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0050] Term description:

[0051] The TRAF2 protein refers to tumor necrosis factor receptor-associated factor 2;

[0052] The PERK protein refers to protein kinase R-like endoplasmic reticulum kinase;

[0053] The Caspase-3 protein refers to cysteine aspartic acid protease 3;

[0054] The DAXX protein refers to death domain-associated protein;

[0055] The MKK3 protein refers to mitogen-activated protein kinase kinase 3;

[0056] The p38 protein refers to mitogen-activated protein kinase;

[0057] The CyclinE protein refers to cyclin E.

[0058] The parent nuclear structural formula of Chiisanoside and its analogs referred to in the present invention is as follows:

[0059]

[0060] Among them, R1 is selected from -H, -β-D-Glc, -β-D-Glc-(6←1)-β-D-Glc, and -β-D-Glc-(6←1)-β-D-Glc-(6←1)-α-L-Rha;

[0061] R2 is selected from -H, -OH, and -O(CH2)2CH3;

[0062] R3 is selected from -CH3 and -CH2OH;

[0063] R4 is selected from -OH and -H;

[0064] R5 is selected from -CH(CH2)CH3, -C(O)CH3, and -CH(CH3)COOH.

[0065] Each monomer compound used in the following examples is extracted from the leaves (or fruits) of Eleutherococcus sessiliflorus (Rupr.) Maxim. (or Eleutherococcus senticosus Rupr.), and their structural and content information is shown in Table 1. The extraction and separation process is briefly described as follows:

[0066] 1. Using the leaves of Eleutherococcus sessiliflorus (Rupr.) Maxim. as raw materials

[0067] Extraction and enrichment of total saponins: 5 kg of the leaves of Eleutherococcus sessiliflorus (Rupr.) Maxim. (collected in mid-July, blanched and dried) were ultrasonically extracted 3 times with 10 times the amount (volume-mass ratio) of 70% ethanol-water. The extraction solutions were combined, and the ethanol was recovered until there was no alcohol smell. The solution was suspended on a D101 macroporous adsorption resin column, 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 recovered to obtain the 50% ethanol elution fraction (reserved), and eluted with 90% ethanol-water until the effluent was colorless and the eluate was collected and recovered to obtain the 90% ethanol elution fraction (reserved).

[0068] Separation of 50% ethanol elution fraction: Take the 50% ethanol elution fraction, load it onto a silica gel column by dry method (upward volume - silica gel = 1:20), elute with chloroform - methanol (20:1), monitor by silica gel thin - layer chromatography until no fraction is eluted; elute with chloroform - methanol (5:1), monitor by silica gel thin - layer chromatography until no fraction is eluted, recover the solvent to obtain the Y - Fr - 5 - 1 fraction; elute with chloroform - methanol (3:1), monitor by silica gel thin - layer chromatography until no fraction is eluted, recover the solvent to obtain the Y - Fr - 3 - 1 fraction; elute with chloroform - methanol (1:1), monitor by silica gel thin - layer chromatography until no fraction is eluted, recover the solvent to obtain the Y - Fr - 1 - 1 fraction.

[0069] Separation of Y - Fr - 5 - 1 fraction: Dissolve the Y - Fr - 5 - 1 fraction in methanol, separate by C18 reversed - phase high - performance liquid chromatography, detect at 205 nm, elute with a gradient of acetonitrile - water (18:82 - 32:68), collect the fractions at the corresponding peak positions according to the chromatogram, recover the solvent, and recrystallize with methanol to obtain the main component Chiisanoside.

[0070] Separation of Y - Fr - 3 - 1 fraction: Dissolve the Y - Fr - 3 - 1 fraction in methanol, separate by C18 reversed - phase high - performance liquid chromatography, detect at 205 nm, elute with a gradient of acetonitrile - water (24:76 - 38:62), collect the fractions at the corresponding peak positions according to the chromatogram, recover the solvents respectively, and recrystallize with methanol to obtain the main components Divaroside and Sessiloside A1 in sequence.

[0071] Separation of Y - Fr - 1 - 1 fraction: Dissolve the Y - Fr - 1 - 1 fraction in methanol, separate by C18 reversed - phase high - performance liquid chromatography, detect at 205 nm, elute with a gradient of acetonitrile - water (33:67 - 41:59), collect the fractions at the corresponding peak positions according to the chromatogram, recover the solvent, and recrystallize with methanol to obtain the main component Chiisanogenin.

[0072] 2. Using Acanthopanax senticosus fruits as raw materials

[0073] Extraction and enrichment of total saponins: 5 kg of Acanthopanax senticosus fruits (collected at the end of September and dried), extract 3 times with 10 times the amount (volume - mass ratio) of 70% ethanol - water by ultrasonic method, combine the extracts, recover ethanol until there is no alcohol smell, suspend and load onto a D101 macroporous adsorption resin column, wash with water until the effluent is colorless, elute with 30% ethanol until the effluent is colorless, elute with 50% ethanol - water until the effluent is colorless and collect the eluate, recover and dissolve to obtain the 50% ethanol elution fraction (for standby), elute with 90% ethanol - water until the effluent is colorless and collect the eluate, recover and dissolve to obtain the 90% ethanol elution fraction (for standby).

[0074] Separation of 50% ethanol elution fraction: Take the 50% ethanol elution fraction, load it onto a silica gel column by dry method (sample amount - silica gel = 1:20), elute with chloroform - methanol (20:1), monitor by silica gel thin - layer chromatography until no fractions are eluted; then elute with chloroform - methanol (5:1), monitor by silica gel thin - layer chromatography until no fractions are eluted, and recover the solvent to obtain the G - Fr - 5 - 1 fraction; elute with chloroform - methanol (3:1), monitor by silica gel thin - layer chromatography until no fractions are eluted, and recover the solvent to obtain the G - Fr - 3 - 1 fraction; elute with chloroform - methanol (1:1), monitor by silica gel thin - layer chromatography until no fractions are eluted, and recover the solvent to obtain the G - Fr - 1 - 1 fraction.

[0075] Separation of G - Fr - 5 - 1 fraction: Dissolve the G - Fr - 5 - 1 fraction in methanol, separate it by C18 reversed - phase high - performance liquid chromatography, detect at 205 nm, elute with an acetonitrile - water (18:82 - 32:68) gradient, collect the fractions at the corresponding peak positions according to the chromatogram, recover the solvent respectively, and recrystallize with methanol to obtain the main components 24 - Hydroxychiisanoside, 22α - Hydroxychiisanoside, Acanthosessilioside K, Chiisanoside, 1 - Deoxychiisanoside.

[0076] Separation of G - Fr - 3 - 1 fraction: Dissolve the G - Fr - 3 - 1 fraction in methanol, separate it by C18 reversed - phase high - performance liquid chromatography, detect at 205 nm, elute with an acetonitrile - water (24:76 - 38:62) gradient, collect the fractions at the corresponding peak positions according to the chromatogram, recover the solvent, and recrystallize with methanol to obtain the main component Acanthosessilioside F successively.

[0077] Separation of G - Fr - 1 - 1 fraction: Dissolve the G - Fr - 1 - 1 fraction in methanol, separate it by C18 reversed - phase high - performance liquid chromatography, detect at 205 nm, elute with an acetonitrile - water (33:67 - 41:59) gradient, collect the fractions at the corresponding peak positions according to the chromatogram, recover the solvent respectively, and recrystallize with methanol to obtain the main components Elesesterpene F, 22α - Hydroxychiisanogenin, Elesesterpene G, Chiisanogenin.

[0078] Table 1 Structural and content information table of each monomer compound

[0079]

[0080] For each monomer compound obtained by the above separation, through NMR and MS data analysis, its structural data is determined as follows:

[0081] Chiisanoside (abbreviated as CSS in English), white crystal, C48 H 74 O 19 。MS: [M] + 954.4815. 13 C NMR (100 MHz, MeOD) δ ppm: 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。

[0082] Divaroside (abbreviation DVS), white crystal, C 42 H 64 O 15 。MS: [M] + 808.4268. 13 C NMR (100 MHz, MeOD) δ ppm: 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。

[0083] Sessiloside A1 (abbreviation SSA), white crystal, C 36 H 54 O 10 。MS: [M] + 646.3728. 1313C NMR (100 MHz, MeOD) δ ppm: 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.

[0084] Chiisanogenin (abbreviated as CSG), white crystal, C 30 H 44 O5. MS: [M] + 484.3169. 13 13C NMR (100 MHz, CDCl3) δ ppm: 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.

[0085] 24-Hydroxychiisanoside (abbreviated as 24-HCS), white crystal, C 48 H 74 O 20 . MS: [M] + 970.4753. 1313C NMR (100 MHz, Pyridine-d5) δ ppm: 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, 13.83.

[0086] 22α-Hydroxychiisanoside (abbreviated as 22α-HCS), white powder, C 48 H 74 O 20 . MS: [M] + 970.4788. 13 13C NMR (100 MHz, Pyridine-d5) δ ppm: 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, 13.83.

[0087] 22α-Hydroxychiisanogenin (abbreviated as 22α-HCG), white powder, C 30 H 44 O6. MS: [M] + 500.3126. 1313C NMR (100 MHz, Pyridine-d5) δ ppm: 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.

[0088] 1-Deoxychiisanoside (abbreviated as 1-DCS), white powder, C 48 H 74 O 18 . MS: [M] + 938.4875. 13 13C NMR (100 MHz, Pyridine-d5) δ ppm: 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.

[0089] Elesesterpene F (abbreviated as ESP F), white powder, C 30 H 44 O7. MS: [M] + 532.3059. 1313C NMR (100 MHz, Pyridine-d5) δ ppm: 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, 42.17, 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.

[0090] Acanthosessilioside F (abbreviated as ASS F), white powder, C 36 H 54 O 11 .. MS: [M] + 662.3751. 13 13C NMR (100 MHz, Pyridine-d5) δ ppm: 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.

[0091] Elesesterpene G (abbreviated as ESP G), white powder, C 29 H 42 O6. MS: [M] + 486.3058. 13 13C NMR (100 MHz, Pyridine-d5) δ ppm: 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, 42.17, 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.

[0092] Acanthosessilioside K (abbreviated as ASS K in English), white powder, C 51 H 80 O 20 。MS: [M] + 1012.5521. 13 C NMR (100 MHz, Pyridine-d5) δ ppm: 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.

[0093] Unless otherwise specified, other materials and reagents can be obtained through regular purchases.

[0094] Research has found that long-term administration of high doses of D-galactose can induce senescence symptoms in animals, and D-galactose is often used to establish senescence animal models. During the metabolic process, D-galactose will produce excessive ROS, leading to oxidative stress, which in turn causes apoptosis. Oxidative stress can damage biomolecules inside cells, such as DNA, proteins, and lipids, etc., thus accelerating cell senescence. D-galactose can participate in the regulation of senescence by affecting the expression of certain genes. D-galactose can activate MKK3, and then activate p38. Activated p38 can phosphorylate p53, enhancing the stability and transcriptional activity of p53, thereby inducing the expression of the cell cycle arrest-related gene p21, causing cells to arrest in the G1 phase. By using D-galactose to establish a model, a sensorineural presbycusis model can be replicated. Therefore, in this example, D-galactose is used to induce cochlear hair cells or mice to establish a senescence model.

[0095] The Chiisanoside (CSS) and its analogs used in the examples of the present invention were prepared in the laboratory, and their purities were all greater than 98%, and the specific identification data are as shown above.

[0096] Example 1 CSS and its analogs resist D-galactose-induced cochlear hair cell senescence by inhibiting apoptosis

[0097] I. Experimental Methods

[0098] 1. Experimental cell lines

[0099] Inner ear hair cells (HEI-OC1) were donated by the Third Hospital of Jilin University.

[0100] 2. Effects of Chiisanoside (CSS) and its analogs on the viability of HEI-OC1 cells

[0101] Cells were cultured by conventional methods, and the CCK-8 method was used to detect the effects of different concentrations of Chiisanoside (CSS) and its analogs (6.25, 12.5, 25, 50, 100 μM) on the viability of HEI-OC1 cells.

[0102] 3. Effects of Chiisanoside (CSS) and its analogs on the cell viability after D-galactose-induced senescence of inner ear hair cells

[0103] Cells were cultured by conventional methods. After 24 h, D-galactose (60 mg / mL, determined according to experimental exploration) was used to induce cell senescence. After 12 h, different concentrations of Chiisanoside (CSS) and its analogs (6.25, 12.5, 25, 50, 100 μM) were administered for treatment. After 24 h, the CCK-8 method was used to measure the viability of HEI-OC1 cells.

[0104] 4. Crystal violet staining

[0105] HEI-OC1 cells were seeded into 24-well plates at 5×10 4 cells and incubated at 37 °C and 5% carbon dioxide for 24 h. A senescence model was established using 60 mg / mL D-galactose (Yuanye Bio-Technology, purity ≥99.0%). After 12 h, the cells were treated with the medium of the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM) for 24 h, then rinsed twice with PBS and fixed with fixative for 30 minutes, and air-dried appropriately. After staining with 0.1% crystal violet for 20 min, the crystal violet staining solution was aspirated, rinsed twice with PBS, and the cell number and morphology were observed under a fluorescence microscope.

[0106] 5. Live / dead cell analysis

[0107] HEI-OC1 cells were seeded at 5×10 4The density of the cells was inoculated into 24-well plates, and an aging model was established using D-galactose (60 mg / mL). After 12 h, the cells were treated with media containing the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM). After 24 h, the cells were stained with FDA (0.02 mg / mL) and PI (0.02 mg / mL) dyes, incubated in the dark for 10 minutes, and then observed under a fluorescence microscope.

[0108] 6. Flow cytometry analysis of apoptosis

[0109] The cells were inoculated into 6-well plates at a density of 5×10 4 cells per well and cultured for about 24 h after attachment. An aging model was established using D-galactose (60 mg / mL). After 12 h, the cells were treated with media containing the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM), while the cells in the model group were not treated with drugs. At the same time, a blank group and a combined treatment group of Chiisanoside (CSS, 25 μM) and shTRAF2 (the shTRAF2 gene overexpression plasmid and adenovirus were both designed and provided by Shanghai Heyuan Biotechnology Co., Ltd.) were established. After 24 h, the cells were washed, and 400 μL of Annexin V binding solution was added to each cell sample tube to suspend the cells. Then, 5 μL of AnnexinV-FITC staining solution was added and incubated (in the dark, for 15 min). Subsequently, 10 μL of PI (propidium iodide) staining solution was added and incubated continuously (in the dark, for 5 min). The cell suspension was filtered through a 200-mesh filter into a flow tube and immediately detected using a flow cytometer. The entire detection process should be completed within 1 h to avoid fluorescence quenching. Subsequently, the data were analyzed using FlowJo10 software.

[0110] 7. Transcriptomic sequencing

[0111] HEI-OC1 cells were inoculated into 6-well plates at a density of 1×10 6 cells per well for 24 h. An aging model was established using D-galactose (60 mg / mL). After 12 h, the cells were treated with media containing the representative compound Chiisanoside (CSS, 25 μM), while the cells in the model group were not treated with drugs. At the same time, a blank group was established, and the following operations were carried out.

[0112] 7.1 RNA extraction and identification

[0113] After establishing the aging model with D-galactose and treating with the test drug for 24 hours, total RNA was extracted from HEI-OC1 cells using TRIzol reagent. The RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).

[0114] 7.2 qRT-PCR

[0115] Total RNA extracted by the Trizol method from each group was measured for concentration and then reverse transcribed into cDNA according to the instructions of the reverse transcription kit. The reaction system was prepared according to the instructions, and cDNA amplification and detection were performed using a qRT-PCR instrument. The relative expression level of the target gene was calculated based on the value of the cycle threshold (Ct).

[0116] 8. ROS content analysis

[0117] HEI-OC1 cells were seeded in 24-well plates at a density of 5×10 4 per well. After 24 h, an aging model was established using D-galactose (60 mg / mL). After 12 h, the cells were treated with the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM) and the medium containing Chiisanoside (CSS, 25 μM) combined with shTRAF2 for 24 h. Then, the content of reactive oxygen species in HEI-OC1 cells was detected using an active oxygen detection kit with the fluorescent probes DCFH-DA and DAPI.

[0118] 9. Mitochondrial membrane potential detection

[0119] HEI-OC1 cells were seeded in 24-well plates at a density of 5×10 4 per well. After 24 h, an aging model was established using D-galactose (60 mg / mL). After 12 h, the cells were treated with the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM) and the medium containing Chiisanoside (CSS, 25 μM) combined with shTRAF2 for 24 h. Then, JC-1 was used to detect the mitochondrial membrane potential (ΔΨm) in the cells. When the mitochondrial membrane potential is high, the JC-1 probe emits red fluorescence; when the mitochondrial membrane potential is too low, the JC-1 probe emits green fluorescence. Observation and photography were performed using a fluorescence microscope, and the excitation wavelengths of red fluorescence and green fluorescence are 530 and 580 nm, respectively.

[0120] 10. Detection of related biochemical indicators

[0121] The activities of LDH, HMBG-1, MDA, GSH, SOD, and Ca 2+ were measured. HEI-OC1 cells were cultured in 6-well plates (15×10 4 cells / well) and treated according to the previously described protocol. Subsequently, the cells were collected, and the culture medium was used to detect LDH and HMBG-1. After cell lysis, the cells were used to detect MDA, GSH, SOD, and Ca 2+ .

[0122] 11. Analysis of the expression levels of related proteins

[0123] HEI-OC1 cells were seeded in 6-well plates and pretreated with the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM) and Chiisanoside (CSS, 25 μM) combined with shTRAF2 for 24 h. After discarding the culture medium, the cells were washed three times with pre-cooled PBS, then 200 μL of cell lysis buffer was added. After the cells were completely lysed, the cells were scraped off with a pipette tip and aspirated into a sterile 1.5 mL centrifuge tube. The mixture was centrifuged at 12,500 rpm for 25 min at 4 °C, the precipitate was removed, and the supernatant was collected and stored at -80 °C (the cell lysis process should be carried out on an ice box).

[0124] The BCA kit was used to detect the protein concentration of each sample.

[0125] TRAF2, PERK, and Caspase-3 protein content detection kits were used to detect the protein expression levels. After protein quantification by the BCA method, SDS-PAGE gel electrophoresis, membrane transfer, and blocking were performed. The corresponding primary and secondary antibodies were added in sequence, and after adding the luminescent solution, the film was developed on a developer. Using β-actin as a control, Image J software was used to analyze the protein blot.

[0126] 12. Statistical analysis

[0127] All data were statistically analyzed using Prism 9 (GraphPad) statistical software. The data were expressed as Mean ± SD, and the t-test combined with one-way ANOVA was used for comparison between groups.

[0128] II. Experimental results

[0129] 1. Effects of Chiisanoside (CSS) and its analogs on the viability of hair cells

[0130] By measurement, Chiisanoside (CSS) and its analogs (6.25, 12.5, 25, 50, 100 μM) of the present invention did not show obvious cytotoxic activity against HEI-OC1 cells and showed certain proliferative activity. The results are shown in Figure 1 .

[0131] 2. Protective activity of Chiisanoside (CSS) and its analogs against D-galactose-induced senescent hair cells

[0132] In terms of the cytoprotective activity of HEI-OC1, Chiisanoside (CSS) and its analogues all showed good protective effects on the D-galactose-induced cochlear hair cell senescence model. Among them, Chiisanoside (CSS), Divaroside (DVS), Sessiloside A1 (SSA) and Chiisanogenin (CSG) had better activities. 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 , and the EC 50 value is shown in Table 2.

[0133] Table 2 EC 50 values (μM) of CSS and its analogues on the D-galactose-induced cochlear hair cell senescence model

[0134] Compound <![CDATA[EC 50 (μM)]]> Compound <![CDATA[EC 50 (μM)]]> CSS 7.61 CSG 19.46 DVS 14.37 SSA 8.62 ESPF 31.26 ESPG 21.89 ASSF 28.32 ASSK 35.46 1-DCS 30.46 24-HCS 29.56 22α-HCS 36.41 22α-HCG 25.32

[0135] Subsequent experiments were all carried out with Chiisanoside (CSS) as the representative. Divaroside (DVS), Sessiloside A1 (SSA) and Chiisanogenin (CSG) had similar effects to CSS.

[0136] 3. Effects of Chiisanoside (CSS) on the morphology of HEI-OC1 cells

[0137] 3.1 Crystal violet staining

[0138] The crystal violet staining results of each component on HEI-OC1 cells are shown in Figure 3 . It was observed that the number of viable HEI-OC1 cells in the model group was significantly lower than that in the control group, and the cytoskeleton was significantly damaged and deformed. In contrast, the viability of cells in the Chiisanoside (CSS) pretreatment group, especially the Chiisanoside (CSS, 25 μM) pretreatment group, was significantly improved, and there was an obvious reversal of the significant damage to the cell morphology compared with the model group. The cell arrangement was relatively neat and the morphology was regular.

[0139] 3.2 Live / dead cell analysis

[0140] The results of double staining of each component on HEI-OC1 cells with fluorescein diacetate (FDA) and propidium iodide (PI) are shown in Figure 4。It was observed that very few PI (red)-stained dead cells were observed in the blank group, while a significantly increased number of dead cells stained with PI (red) were observed in the model group. Pretreatment with Chiisanoside (CSS), especially pretreatment with Chiisanoside (CSS, 25 μM), significantly increased the uptake of FDA (green) by live cells and decreased the PI staining (red) of dead cells.

[0141] 3.3 Effects of Chiisanoside (CSS) on apoptosis of HEI-OC1 cells

[0142] Flow cytometry analysis was as Figure 5 shown. After D-galactose treatment, the percentage of apoptosis and necrosis of HEI-OC1 cells increased significantly to 49.14% (P < 0.01 compared with control). Compared with the model group, after treatment with 12.5, 25, and 50 μM of Chiisanoside (CSS), the apoptosis and necrosis rates decreased to 37.34% (P < 0.05), 13.72% (P < 0.01), and 28.45% (P < 0.01), respectively. However, shTRAF2 blocked the effect of Chiisanoside (CSS, 25 μM). In summary, it was demonstrated that Chiisanoside (CSS) could inhibit apoptosis by reducing the production of TRAF2 in cells and resist the irreversible damage caused by D-galactose, and this protective effect might be related to the activation of the TRAF2-related pathway. These results indicate that Chiisanoside (CSS) can effectively improve D-galactose-induced apoptosis and necrosis of HEI-OC1 cells and play a pre-protective role against D-galactose-induced cochlear hair cell senescence.

[0143] 4. Transcriptome differential analysis

[0144] DEGs in the model group (M) and Chiisanoside group (CSS) were enriched by KEGG pathways. As Figure 6As shown, the top 20 pathways with the most reliable enrichment significance (i.e., the smallest Q value) were selected to present the results. Among them, the present invention selected the most relevant and significantly different apoptosis, cellular senescence, cell cycle, and MAPK signaling pathways. Through gene expression differences, it was found that Chiisanoside (CSS) could significantly counteract the changes in the expression levels of genes related to the above pathways, such as TRAF2, PERK, Caspase-3, DAXX, MKK3, p38, and CyclinE, induced by D-galactose-induced senescence. Especially Chiisanoside (CSS, 25 μM), and the results are as Figure 7 shown.

[0145] 5. ROS content analysis

[0146] The results are as Figure 8 shown. Compared with the blank group, the fluorescence intensity of the D-galactose treatment group (model group) was significantly enhanced (P < 0.01). Compared with the model group, the ROS fluorescence in the Chiisanoside (CSS) group was significantly weakened, especially Chiisanoside (CSS, 25 μM). shTRAF2 significantly blocked the effect of Chiisanoside (CSS, 25 μM), further indicating that Chiisanoside (CSS, 25 μM) reversed the production of ROS in HEI-OC1 cells induced by D-galactose and was related to its effect on TRAF2.

[0147] 6. Mitochondrial membrane potential staining

[0148] The oxidation-sensitive dyes DCFH-DA and JC-1 probes were used to detect the mitochondrial membrane potential δψm in cells. As Figure 9 shown, compared with the control group, the fluorescence red / green ratio of the D-galactose treatment group (model group) was significantly weakened (P < 0.01), indicating that the excessive production of ROS in HEI-OC1 cells led to an increase in mitochondrial membrane potential. On the contrary, pretreatment with Chiisanoside (CSS) significantly enhanced the fluorescence red / green ratio, especially Chiisanoside (CSS, 25 μM). And shTRAF2 significantly blocked the effect of Chiisanoside (CSS, 25 μM), further indicating that the change in mitochondrial membrane potential by Chiisanoside (CSS, 25 μM) was related to its effect on TRAF2.

[0149] 7. Biochemical index detection

[0150] As Figure 10As shown, compared with the control group, the levels of Ca 2+ , MDA, LDH, and HMBG-1 exposure in the D-galactose treatment group (model group) were significantly increased, while the levels of SOD and GSH exposure were significantly decreased (P<0.01). In the Chiisanoside (CSS) pretreatment group, with the increase in dose, the levels of Ca 2+ , MDA, LDH, and HMBG-1 exposure gradually decreased, and the levels of SOD and GSH exposure were significantly increased. In particular, the pretreatment effect with 25 μM Chiisanoside was the most significant. shTRAF2 could reverse the above effects of Chiisanoside (CSS), further demonstrating that the changes in the above cell senescence-related biochemical indexes by Chiisanoside (CSS, 25 μM) were related to the effect on TRAF2.

[0151] 8. Determination of related protein expression

[0152] The results of verifying the activities of related proteins by Western blotting are as Figure 11 shown. Compared with the blank group, in the senescence model induced by D-galactose in HEI-OC1, the protein expression levels of TRAF2, PERK, and Caspase-3 were all significantly increased (P<0.01). Compared with the model group, each group of Chiisanoside (CSS) significantly reduced the expression of the above proteins, and the effect of Chiisanoside (CSS, 25 μM) was the most significant. Further using shTRAF2 reversed the above effect of Chiisanoside (CSS, 25 μM).

[0153] The above experimental results of this example show that Chiisanoside (CSS) and its analogs have a significant effect of resisting D-galactose-induced hair cell senescence and reducing apoptosis, among which Chiisanoside (CSS, 25 μM) is the most significant, and its anti-hair cell senescence and inhibitory effect on hair cell apoptosis are achieved by inhibiting the expression of TRAF2, PERK, and Caspase-3 proteins.

[0154] Example 2 CSS and its analogs resist D-galactose-induced hair cell senescence by inhibiting cell cycle arrest

[0155] I. Experimental method

[0156] 1. Flow cytometry analysis of cell cycle

[0157] In this example, the PI staining method was used to detect the cell cycle distribution. HEI-OC1 cells were seeded at 1×10 6Cells were seeded at a density of [number] cells / well in 6-well plates for 24 h. An aging model was established using D-galactose (60 mg / mL). After 12 h, the cells were treated with media containing the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM). Meanwhile, a co-treatment group of Chiisanoside (CSS, 25 μM) and shDAXX (the shDAXX gene overexpression plasmid and adenovirus were designed and provided by Shanghai Heyuan Biotechnology Co., Ltd.) was established. After 24 h, the cells were stained with PI solution (50 μg / mL), incubated in the dark for 30 min, and the cell suspension was filtered through a 200-mesh filter into flow cytometry tubes. Subsequently, the cell cycle distribution was immediately detected using a flow cytometer. The entire detection process should be completed within 1 h to avoid fluorescence quenching.

[0158] 2. β-Galactosidase Staining

[0159] β-Galactosidase staining was performed using a β-galactosidase staining kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0160] HEI-OC1 cells were seeded at a density of [number] 6 cells / well in 6-well plates for 24 h. An aging model was established using D-galactose (60 mg / mL). After 12 h, the cells were treated with media containing the representative compound Chiisanoside (CSS, 12.5, 25, 50 μM). Meanwhile, a co-treatment group of Chiisanoside (CSS, 25 μM) and shDAXX (the shDAXX gene overexpression plasmid and adenovirus were designed and provided by Shanghai Heyuan Biotechnology Co., Ltd.) was established. After 24 h, the cells were fixed with fixative at room temperature for 15 min, washed 3 times with PBS, 500 μL of working solution (consisting of Solution A, Solution B, Solution C, and X-Gal) was added to each well, and the cells were incubated overnight in an oven at 37°C (without CO2). After 12 h, the cells were observed and photographed under an inverted fluorescence microscope.

[0161] 3. Analysis of Protein Expression Levels

[0162] Total cellular proteins were extracted as in Example 1. The protein concentration of each sample was detected using a BCA kit (Shanghai Beyotime Biotechnology Co., Ltd.). Protein expression levels were detected using DAXX, MKK3, p38, and CyclinE protein content detection kits (Shanghai Beyotime Biotechnology Co., Ltd.). After protein quantification by the BCA method, SDS-PAGE gel electrophoresis, membrane transfer, and blocking were performed. The corresponding primary and secondary antibodies were added sequentially. After adding the luminescent solution, the film was developed on a developer. Using β-actin as a control, protein blots were analyzed using Image J software.

[0163] 4. Statistical Analysis

[0164] All data were statistically analyzed using Prism 9 (GraphPad) statistical software. The data are expressed as Mean±SD, and the comparison between groups was performed using t-test combined with one-way ANOVA.

[0165] II. Experimental Results

[0166] 1. Detection of cell cycle by flow cytometry

[0167] The cell cycle was blocked in the G1 phase, and the cells senesced. Flow cytometry was used to detect the cell cycle arrest of HEI-OC1 cells in each group. The results are as Figure 12 shown. Compared with the control group, the proportion of G1-phase cells in the D-galactose group (model group) increased, and the proportion of G2-phase cells decreased, indicating a G1-phase arrest in the cell cycle. When the cells were treated with Chiisanoside (CSS), this phenomenon was significantly reversed, especially with Chiisanoside (CSS, 25 μM). However, shDAXX blocked the above therapeutic effect of Chiisanoside (CSS, 25 μM). In summary, the experiment demonstrated that Chiisanoside (CSS) could inhibit the cell cycle arrest of cochlear hair cells caused by D-galactose by acting on DAXX.

[0168] 2. Senescence-associated β-galactosidase staining

[0169] Senescence-associated β-galactosidase (SA-β-gal) is an enzyme closely related to cell senescence. SA-β-gal is widely regarded as one of the reliable biomarkers of cell senescence. Its activity is significantly enhanced in senescent cells, while it is low in normal or proliferating cells. By detecting the activity of SA-β-gal in cells, it is possible to intuitively determine whether the cells have entered the senescent state. The experimental results are as Figure 13 shown. After inducing HEI-OC1 cell senescence with D-galactose (model group), the number of SA-β-gal positive cells was significantly higher than that in the control group (P<0.01). Treatment of cochlear hair cells with Chiisanoside (CSS) significantly reversed this phenomenon, especially with Chiisanoside (CSS, 25 μM). However, shDAXX blocked the above therapeutic effect of Chiisanoside (CSS, 25 μM). In summary, the experiment demonstrated that Chiisanoside (CSS) could inhibit the senescence of cochlear hair cells caused by D-galactose by acting on DAXX.

[0170] 3. Determination of related protein expression

[0171] The results are as Figure 14As shown, compared with the blank group, after D-galactose induced the senescence of HEI-OC1 cells (model group), the expression levels of DAXX, p-MKK3, and p-p38 MAPK in cochlear hair cells were significantly increased (P<0.01), and the expression level of CyclinE was significantly decreased (P<0.01), which then became the main reason for D-galactose induced cell cycle arrest and cell senescence of cochlear hair cells HEI-OC1. Compared with the model group, the expression levels of DAXX, p-MKK3, and p-p38 MAPK in the Chiisanoside (CSS) pretreatment group significantly decreased with the increase of dose, and the expression level of CyclinE significantly increased, and reached the peak at Chiisanoside (CSS, 25 μM). And the silencing of the DAXX gene (shDAXX) inhibited the effect of Chiisanoside (CSS, 25 μM) to a certain extent.

[0172] In summary, Chiisanoside (CSS) inhibits the expression of DAXX, p-MKK3, and p-p38 MAPK by regulating the DAXX / MKK3 / p38 / CyclinE signaling pathway, and then loses the blocking effect on CyclinE-CDK2, and promotes the cell cycle to enter the S phase from G1, initiating DNA replication. Thereby inhibiting D-galactose induced cell cycle arrest and cell senescence of cochlear hair cells.

[0173] Example 3 CSS and its analogs resist hearing impairment induced by D-galactose in sensorineural presbycusis mice by inhibiting apoptosis and cell cycle arrest of inner ear cells

[0174] I. Experimental methods

[0175] 1. Experimental animals

[0176] Adult male C57BL / 6 mice, weighing 20-22 g, were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. and raised in a laboratory-controlled environment. Randomly divided into groups, with 8 mice in each group.

[0177] 2. Establish a treatment model for sensorineural presbycusis mice

[0178] Adult male C57BL / 6 mice were selected and divided into five groups of equal number, namely the control group, D-galactose group (D-Gal), high-dose treatment group (D-Gal+CSS-H), medium-dose treatment group (D-Gal+CSS-M), and low-dose treatment group (D-Gal+CSS-L). The control group was intraperitoneally injected with normal saline for eight consecutive weeks; the experimental group was intraperitoneally injected with D-galactose at 600 mg / kg for eight consecutive weeks; after four weeks of intraperitoneal injection of D-galactose at 600 mg / kg, the high-dose group, medium-dose group, and low-dose group were respectively intragastrically administered with the monomer compound of Chiisanoside (CSS) at 50 mg / kg, 25 mg / kg, and 12.5 mg / kg, and then intraperitoneally injected with D-galactose at 600 mg / kg 2 hours later for four consecutive weeks.

[0179] 3. Auditory brainstem response was used to detect the hearing threshold of mice

[0180] Auditory Brainstem Response (ABR) is an important electrophysiological examination method for evaluating the function of the auditory system. By giving specific sound stimuli, such as clicks or tone bursts, to the ears of the subjects, the sound signals are transmitted into the inner ear along the auditory conduction pathway, passed through the auditory nerve to the brainstem, and a series of potential changes related to the sound stimuli will occur in different parts of the brainstem. These potential changes can be recorded by electrodes placed at specific positions on the scalp to form the auditory brainstem response waveform.

[0181] 3.1 ABR audiometry

[0182] After administration, ABR detection was performed on the mice to observe the changes in their hearing. A reference electrode, recording electrode, and ground electrode were inserted subcutaneously behind the ears of the mice, and the speaker was placed on the side of the ear to be measured of the mice to record the waveform and observe the changes in their hearing.

[0183] 3.2 Cochlear basilar membrane spreading and staining

[0184] The cochlear basilar membranes of the model mice were spread and immunofluorescently stained respectively, and the number of hair cells and cilia of the mice were compared with Myosin7A, Phalloidin, and 4',6-diamidino-2-phenylindole (DAPI).

[0185] 4. Determination of related protein expression

[0186] The protein expression levels of TRAF2, PERK, Caspase-3, DAXX, MKK3, p38, CyclinE, etc. in the cochlear tissue homogenates of mice pretreated with the representative drug Chiisanoside (CSS) were detected. Cochlear tissues of mice were taken, lysate was added to extract proteins, and shTRAF2 and shDAXX were respectively added. After protein quantification using the BCA method, SDS-PAGE gel electrophoresis, membrane transfer, and blocking were carried out. The corresponding primary and secondary antibodies were added in sequence. After adding the luminescent solution, imaging was performed on the imaging instrument. Using β-actin as a control, the protein blot was analyzed using Image J software.

[0187] 5. Statistical analysis

[0188] The t-test and analysis of variance were performed using the SPSS statistical software package.

[0189] II. Experimental results

[0190] 1. Hearing detection of D-galactose-induced sensorineural presbycusis mice

[0191] After successful modeling, the hearing of the D-galactose group was monitored, and the results are as Figure 15 shown. Compared with the control group, the hearing threshold of the D-galactose model group mice was significantly increased (P<0.01), with an average of 90 db, confirming that D-galactose-induced presbycusis in mice can lead to severe hearing loss. The hearing threshold of the group pretreated with Chiisanoside (CSS) decreased with the increase of the administration dose and reached the peak at the dose of 50 mg / kg (P<0.01).

[0192] 2. Basilar membrane spreading staining

[0193] By labeling hair cells with Myosin7a and DAPI, it was found that the outer hair cells in the D-galactose injury group (model group) were significantly lost, and the number of hair cells in the basal turn of the cochlea was the most severely damaged. The loss of hair cells in the Chiisanoside (CSS) group was less than that in the D-galactose injury group. The survival rate of hair cells is shown in Figure 16 .

[0194] 3. Expression levels of apoptosis-related proteins

[0195] The results are as Figure 17As shown, compared with the blank group, the protein expression levels of TRAF2, PERK, and Caspase-3 in the cochlea were significantly increased in D-galactose-induced mice. Compared with the model group, the expression levels of TRAF2, PERK, and Caspase-3 in the Chiisanoside (CSS) pretreatment group decreased significantly with the increase of dose and reached the peak at the dose of 50 mg / kg. The above effects of Chiisanoside (CSS) were reversed by TRAF2 gene silencing. This is consistent with the detection results of cell experiments, further strongly indicating that Chiisanoside (CSS) inhibits the protein expression of TRAF2, PERK, and Caspase-3 by regulating the TRAF2 / PERK / Caspase-3 signaling pathway, and inhibits the apoptosis of cochlear hair cells caused by endoplasmic reticulum stress induced by D-galactose, resulting in sensorineural presbycusis in mice.

[0196] 4. Expression levels of cell cycle-related proteins in the cochlea of mice

[0197] The results are as Figure 18 shown. To further determine that Chiisanoside (CSS) plays an important regulatory role in D-galactose-induced cochlear hair cell cycle arrest by regulating the DAXX / MKK3 / p38 / CyclinE signaling pathway. The expression levels of DAXX, p-MKK3, p-p38 MAPK, and CyclinE were evaluated by Western blotting. The results showed that compared with the blank group, the expression levels of DAXX, p-MKK3, and p-p38 MAPK in the cochlea were significantly increased in D-galactose-induced mice, and the expression level of CyclinE was significantly decreased, which was the main reason for D-galactose-induced G1 phase arrest of inner hair cells in the cochlea and sensorineural presbycusis in mice. Compared with the model group, the expression levels of DAXX, p-MKK3, and p-p38 MAPK in the Chiisanoside (CSS) pretreatment group were significantly decreased, and the expression level of CyclinE was significantly increased, also showing a dose-dependent trend. The above effects of Chiisanoside (CSS) were reversed by DAXX gene silencing. These results further indicate that Chiisanoside (CSS) inhibits the protein expression of DAXX, p-MKK3, and p-p38 MAPK by regulating the DAXX / MKK3 / p38 / CyclinE signaling pathway, thereby losing the inhibitory effect on CyclinE-CDK2, promoting the cell cycle to enter the S phase from G1, initiating DNA replication, and thus inhibiting D-galactose-induced cochlear hair cell cycle arrest and cell senescence, and further inhibiting the occurrence of D-galactose-induced sensorineural presbycusis in mice.

[0198] In summary, Chiisanoside (CSS) inhibits the protein expression of TRAF2, PERK, and Caspase-3 by regulating the TRAF2 / PERK / Caspase-3 signaling pathway, thereby inhibiting the apoptosis of cochlear hair cells caused by endoplasmic reticulum stress induced by D-galactose and preventing sensorineural presbycusis in mice. Meanwhile, Chiisanoside (CSS) also regulates the DAXX / MKK3 / p38 / CyclinE signaling pathway, inhibits the protein expression of DAXX, p-MKK3, and p-p38 MAPK, and then loses the inhibitory effect on CyclinE-CDK2, promoting the cell cycle transition from G1 to S phase and initiating DNA replication, thus inhibiting the cell cycle arrest and cellular senescence of cochlear hair cells induced by D-galactose, and further preventing the occurrence of sensorineural presbycusis in mice induced by D-galactose.

[0199] Example 4 Tablet Preparation

[0200] Reagents: Starch (pharmaceutical grade, Tianjin Jindong Tianzheng Fine Chemical Reagent Factory); Citric Acid (Shanghai Macklin Biochemical Co., Ltd.); Magnesium Stearate (Shanghai Macklin Biochemical Co., Ltd.).

[0201] Preparation Method:

[0202] ① Preparation of 10% starch paste: Dissolve 0.25 g of citric acid in 25 mL of pure water, add 2.5 g of starch and disperse evenly, then heat to gelatinize to obtain 10% starch paste.

[0203] ② Granulation: Mix 20 g of Chiisanoside powder with 20 g of starch evenly, then add 10% starch paste and mix well, grind to form a soft material, granulate through a 16-mesh sieve, and dry at 60 °C for 1 h. After sieving with a 16-mesh sieve, add the lubricant magnesium stearate and press into tablets using a shallow punch with a diameter of 10 mm.

[0204] The obtained tablets are off-white in color, with uniform color, consistent thickness, and moderate hardness. The tablet weight and disintegration time meet the requirements. The obtained Chiisanoside tablets meet the requirements and can be used as tablets.

[0205] Example 5 Preparation of Suspension Injection

[0206] Reagents: Polylactic acid (PLA, Shanghai Zhenzhun Biotechnology Co., Ltd.); Poly (lactic-co-glycolic acid) (PLGA, Shanghai Yuanye Biotechnology Co., Ltd.); Poloxamer 188 (Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.); Dichloromethane, Methanol, Acetonitrile, etc. (Tianjin Tiantai Chemical Co., Ltd.).

[0207] Preparation Method:

[0208] ① Preparation of polymer microparticles: Weigh 2.5 g of Chiisanoside and 2.5 g of the carrier (PLA / PLGA) and place them in a 50 mL round-bottom flask. Add 5 mL of dichloromethane to dissolve, and remove most of the organic solvents by distillation under reduced pressure at 28 °C. Then, vacuum dry at 40 °C for 24 h until all the solvents are removed. Crush and pass through a sieve with a pore size of 150 μm to obtain Chiisanoside polymer microparticles.

[0209] ② Preparation of Chiisanoside suspension injection: Disperse 2.5 g of Chiisanoside polymer microparticles in 250 mL of an aqueous solution containing 10 g / L of poloxamer 188 stabilizer under continuous stirring until complete dispersion. Grind the drug dispersion to the required particle size, take out to obtain the Chiisanoside polymer microparticle suspension, and centrifuge at 3000 r·min -1 for 1 min, and disperse with 10 mL of the stabilizer aqueous solution to concentrate the preparation to about 25 g / L.

[0210] The obtained suspension injection has uniform particle size, and both the water content and the surface particle size of the preparation meet the regulations. It has a better in vitro sustained-release effect and good stability. The obtained C suspension injection meets the requirements and can be used as a suspension injection.

[0211] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a triterpenoid saponin and its analogs in the preparation of a medicament for treating presbycusis, characterized in that: The triterpenoid saponin and its analogs are Chiisanoside, Divaroside, Sessiloside A1 or Chiisanogenin.

2. The use according to claim 1, characterized in that: The dosage forms of the drug include oral preparation and injection.

3. The use according to claim 2, characterized in that: The oral dosage form includes tablets.

4. The use according to claim 2, characterized in that: The injection includes a suspension injection.

5. The use according to claim 1, characterized in that: The presbycusis is sensorineural presbycusis.

6. The use according to claim 1, characterized in that: The triterpenoid saponin and its analogs achieve the treatment of presbycusis by inhibiting the aging of ear hair cells.

7. The use according to claim 6, characterized in that: The triterpenoid saponins and analogs thereof inhibit the aging of ear hair cells by inhibiting apoptosis and cell cycle arrest of ear hair cells.

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