CN115463141B - Use of ganglioside GM1 in medicaments for hearing impairment - Google Patents
Use of ganglioside GM1 in medicaments for hearing impairment Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention belongs to the technical field of pharmaceutical application. In particular to the application of ganglioside GM1 in preparing a medicament for preventing, relieving or treating hearing impairment. The ganglioside GM1 can directly improve the hearing impairment caused by chemotherapeutics such as platinum drugs, taxol or taxol combined with the platinum drugs, especially the hearing impairment caused by hair cell impairment, thereby effectively improving the life quality of cancer patients after chemotherapy. The ganglioside GM1 also has the effects of preventing, relieving or treating the noise deafness patients, the presbycusis patients and the hearing injury patients caused by the side effect of the medicament, especially the hearing injury caused by the injury of hair cells, so that the life quality of the patients is improved.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical application. In particular to the application of ganglioside GM1 in preparing a medicament for preventing, relieving or treating hearing impairment. The hearing impairment includes hair cell impairment caused by chemotherapeutic drugs, as well as noisy deafness, presbycusis, and hair cell impairment due to drug side effects.
Background
Platinum-based chemotherapeutics are believed to be the primary cause of hearing impairment, including chemotherapy with cisplatin, oxaliplatin, and paclitaxel and their combination with platinum-based drugs (cisplatin or oxaliplatin). Basic chemotherapy drugs and radiotherapy of the head and neck are the most likely treatments for cancers that cause hearing impairment. The auditory system is sensitive to chemotherapeutics, mainly consisting in the unavoidable damage of hair cells caused by platinum drugs such as cisplatin, oxaliplatin, paclitaxel and their combination with platinum-based chemotherapeutics.
In clinical treatment, there is a lack of attention to hearing impairment in tumor chemotherapeutic patients, with a major concern about the efficacy of chemotherapy. Therefore, treatment of the hearing impairment of the tumor patient caused by chemotherapy is not emphasized, and the hearing impairment of the patient is not treated in a large amount. If chemotherapy or radiation therapy is necessary and the patient has developed or is likely to develop hearing loss, then some means of prevention or treatment may be considered.
Currently, the clinical treatment of hearing impairment is mainly to help improve the hearing function of the patient by means of a hearing aid, however, this approach does not fundamentally repair or treat the auditory system of the patient, rather than prevent the occurrence of such hearing impairment beforehand. Currently, there is no available prophylactic, palliative or therapeutic agent for the treatment of hearing impairment in patients with chemotherapy. Therefore, there is a need for a drug that can prevent, alleviate or treat hearing impairment caused by chemotherapy drugs to improve the quality of life of cancer patients after chemotherapy.
Disclosure of Invention
The invention aims at providing a use of ganglioside GM1 in preparing a medicament for preventing, relieving or treating hearing impairment caused by a chemotherapeutic medicament.
In some preferred embodiments, the chemotherapeutic agent of the present invention is selected from the group consisting of platinum, paclitaxel, or any combination thereof; preferably, the chemotherapeutic agent is selected from cisplatin, oxaliplatin, paclitaxel, a combination of cisplatin and paclitaxel, a combination of oxaliplatin and paclitaxel; more preferably, wherein the paclitaxel is albumin-bound paclitaxel.
The use of the invention, in some preferred embodiments, the hearing impairment comprises a varying degree of hair cell impairment in the patient.
In other preferred embodiments, the hair cell damage comprises: the disappearance/decrease of normal cells, the increase of abnormal cells, the abnormality of cell location or cell relative location, the abnormality of characteristic index molecules (such as Myosin7a, etc.) in cells.
The use of the invention, in some preferred embodiments, the patient comprises a oncological patient selected from patients suffering from multiple myeloma, digestive tract tumor, respiratory tract tumor, head and neck tumor, breast cancer, reproductive system tumor, and the like, and other patients suitable for platinum, paclitaxel, and combination chemotherapy thereof, and other solid tumor and hematological tumor patients using various chemoradiotherapy.
In a preferred embodiment of the invention, the ganglioside GM1 is sodium monosialotetrahexosyl ganglioside.
In the invention, the preparation form of ganglioside GM1 is selected from injection preparation, oral preparation, local administration preparation or external preparation; preferably, the ganglioside GM1 is in the form of an injectable formulation.
In some preferred embodiments, the ganglioside GM1 of the present invention is administered before, during or after the administration of the chemotherapeutic agent.
It is also an object of the present invention to provide a method for preventing hearing impairment in a patient suffering from a tumor, comprising administering to the patient a ganglioside GM1 in combination with a chemotherapeutic agent selected from cisplatin, oxaliplatin, paclitaxel, a combination of cisplatin and paclitaxel, or a combination of oxaliplatin and paclitaxel, wherein the hearing impairment comprises hair cell impairment of varying degrees, comprising: the disappearance/decrease of normal cells, the increase of abnormal cells, the abnormality of cell location or cell relative location, the abnormality of characteristic index molecules (such as Myosin7a, etc.) in cells.
It is an object of a third aspect of the present invention to provide a use of ganglioside GM1 in the manufacture of a medicament for preventing, alleviating or treating hearing impairment in a patient, wherein the patient is selected from the group consisting of a patient with noise-induced deafness, a patient with presbycusis, and a patient with hearing impairment due to side effects of the medicament, said hearing impairment comprising hair cell impairment in the patient to varying degrees.
The invention has the following beneficial effects:
1. the ganglioside GM1 can obviously improve the hearing injury caused by platinum drugs, paclitaxel or paclitaxel combined with platinum drug chemotherapy (especially the hearing injury caused by hair cell injury), and can prevent, relieve or treat the hearing injury caused by chemotherapy drugs of patients, thereby effectively improving the life quality of cancer patients after chemotherapy.
2. Experimental data show that compared with a blank control group, the ganglioside GM1 of the invention has no obvious adverse reaction to animals and good safety.
3. The ganglioside GM has the effects of preventing, relieving or treating the noise deafness patients, the presbycusis patients and the hearing injury patients caused by the side effect of the medicament, especially the hearing injury caused by the injury of hair cells, so that the life quality of the patients is improved.
Drawings
Hpf (hour post fertilization) in the context of the present invention refers to the few hours after fertilization (zebra fish); regarding the significance of the differences between groups: ns (no significant) it is meant that GM1 treated group was not significantly different from control group, p < 0.05; * P < 0.01; * P < 0.001; * P < 0.0001.
FIG. 1 is a graph showing that sodium tetrahexosyl ganglioside monosiale (GM 1) improves cisplatin damage to cochlear hair cell line House Ear Institute-Organ of Corti 1 (HEI-OC 1) cells; wherein, FIG. 1A is a drug-to-HEI-OC 1 cell processing procedure; FIGS. 1B-1D show the pre-protective effect of 0.01 mM-1 mM GM1 on 0.01 mM-1 mM cisplatin-damaged HEI-OC1 cells, respectively; in the figure, p <0.001 for the # cisplatin-damaged group compared to the control group, and p <0.001 for the GM 1-treated group compared to the cisplatin-damaged group.
FIG. 2 is a graph showing that sodium monosialotetrahexosyl ganglioside (GM 1) improves cisplatin damage to zebra fish hair cells; wherein, fig. 2A is a drug-to-transgenic zebra fish treatment procedure; FIGS. 2B and 2C show the pre-protection effect of 0.1-2 mg/ml GM1 on 0.01-0.5 mg/ml cisplatin-damaged zebra fish hair cells at 24hpf treatment; in the figure, ns GM1 treated groups were not significantly different from control groups, and GM1 treated groups were p <0.001 compared to cisplatin treated groups.
FIG. 3 is a graph showing that sodium monosialotetrahexosyl ganglioside (GM 1) improves cisplatin damage to zebra fish hair cells; wherein, FIG. 3A is a treatment program of GM1 and cisplatin to transgenic zebra fish; FIGS. 3B and 3C show the pre-protection effect of 0.1-2 mg/ml GM1 on cisplatin-damaged zebra fish hair cells at 0.01-0.5 mg/ml in 8hpf treatment; in the figure, ns GM1 treated groups were not significantly different from control groups, and GM1 treated groups were p <0.001 compared to cisplatin treated groups.
FIG. 4 is a graph showing that sodium tetrahexosyl ganglioside monosiale (GM 1) improves oxaliplatin damage to cochlear hair cell line House Ear Institute-Organ of Corti 1 (HEI-OC 1) cells; wherein, FIG. 4A is a drug-to-HEI-OC 1 cell processing procedure; FIGS. 4B-4D show the pre-protective effect of 0.01 mM-1 mM GM1 on 0.1 mM-1.5 mM oxaliplatin injured HEI-OC1 cells, respectively; in the figure, p <0.001 for the # # oxaliplatin-damaged group compared to the control group, no significant difference was observed between the ns GM1-treated group and the oxaliplatin-damaged group, and p <0.001 for the GM1-treated group compared to the oxaliplatin-damaged group.
FIG. 5 is a graph showing that sodium monosialotetrahexosyl ganglioside (GM 1) improves the damage of oxaliplatin to zebra fish hair cells; wherein, FIG. 5A is a drug-to-transgenic zebra fish treatment procedure, GM1 pre-protecting zebra fish embryos at 24 hpf; FIGS. 5B and 5C show the pre-protection effect of 0.1-1 mg/ml oxaliplatin on the damaged zebra fish hair cells by 0.1-2 mg/ml GM1 treated with 24 hpf; in the figure, ns GM1 treated group was not significantly different from control group, p <0.001 in GM1 treated group compared to oxaliplatin treated group;
FIG. 6 is a graph showing that sodium monosialotetrahexosyl ganglioside (GM 1) improves the damage of oxaliplatin to zebra fish hair cells; wherein, FIG. 6A is a drug-to-transgenic zebra fish treatment procedure, GM1 pre-protecting zebra fish embryos at 8 hpf; FIGS. 6B and 6C show the pre-protective effect of 0.1mg/ml to 2mg/ml GM1 on oxaliplatin-damaged zebra fish hair cells at 8hpf treatment of 0.1mg/ml to 1mg/ml, respectively; in the figure, ns GM1 treated group was not significantly different from control group, p <0.001 in GM1 treated group compared to oxaliplatin treated group;
FIG. 7 is a graph showing that sodium monosialotetrahexosyl ganglioside (GM 1) improves the damage of albumin-bound paclitaxel to zebra fish hair cells; wherein, FIG. 7A is a drug-to-transgenic zebra fish treatment procedure, GM1 pre-protecting zebra fish embryos at 24 hpf; FIGS. 7B and 7C show the pre-protection effect of 0.1-2 mg/ml GM1 on 0.01-1 mg/ml albumin-bound paclitaxel injured zebra fish hair cells; in the figure, ns GM1 treated group was not significantly different from control group, p <0.001 in GM1 treated group compared to albumin-bound paclitaxel treated group;
FIG. 8 is a graph showing that sodium monosialotetrahexosyl ganglioside (GM 1) improves the damage of albumin-bound paclitaxel to zebra fish hair cells; FIG. 8A is a drug-to-transgenic zebra fish treatment procedure with GM1 pre-protecting zebra fish embryos at 8 hpf; FIGS. 8B and 8C show the pre-protection effect of 0.1-2 mg/ml GM1 on 0.01-1 mg/ml albumin-bound paclitaxel injured zebra fish hair cells; in the figure, ns GM1 treated group was not significantly different from control group, p <0.001 in GM1 treated group compared to albumin-bound paclitaxel treated group;
FIG. 9 is a graph showing that sodium monosialotetrahexosyl ganglioside (GM 1) improves the damage to zebra fish hair cells by albumin-bound paclitaxel in combination with cisplatin; FIG. 9A is a drug-to-transgenic zebra fish treatment procedure with GM1 pre-protecting zebra fish embryos at 8 hpf; FIGS. 9B and 9C show the pre-protective effect of 0.1 to 2mg/ml GM1 on 0.01 to 1mg/ml albumin-bound paclitaxel and 0.01 to 0.5mg/ml cisplatin-injured zebra fish hair cells, respectively; in the figure, ns GM1 treated group was not significantly different from control group, p <0.001 compared to albumin-bound paclitaxel-combined cisplatin treated group;
FIG. 10 is a graph showing that sodium monosialotetrahexosyl ganglioside (GM 1) improves the damage to zebra fish hair cells by albumin-bound paclitaxel in combination with oxaliplatin; wherein, fig. 10A is a drug-to-transgenic zebra fish treatment procedure, GM1 pre-protecting zebra fish embryos at 8 hpf; FIGS. 10B and 10C show the pre-protective effect of GM1 in the range of 0.1mg/ml to 2mg/ml on the damage of oxaliplatin to zebra fish hair cells in the range of 0.01mg/ml to 1mg/ml albumin-bound paclitaxel and in the range of 0.1mg/ml to 1mg/ml, respectively; in the figure, ns GM1 treated groups were not significantly different from control groups, and GM1 treated groups were p <0.001 compared to albumin-bound paclitaxel-oxaliplatin treated groups.
FIG. 11 is sodium monosialotetrahexosyl ganglioside (GM 1) improving cisplatin-induced cochlear hair cell injury in mice; wherein, fig. 11A is a view of the mouse cochlea hair cell morphology under a fluorescence microscope; fig. 11B is a hair cell phenotype count showing that cisplatin has significant mouse cochlear hair cytotoxicity, and GM1 pretreatment has significant protective effects, reducing hair cell loss and deformity number.
FIG. 12 is a graph showing the improvement of oxaliplatin-in-combination paclitaxel-induced cochlear hair cell injury in mice by sodium monosialoganglioside (GM 1); wherein, fig. 12A is a view of the mouse cochlea hair cell morphology under a fluorescence microscope; fig. 12B is a hair cell phenotype count showing that oxaliplatin in combination with paclitaxel has significant mouse cochlear hair cytotoxicity, and GM1 pretreatment has significant protective effect, reducing hair cell loss and deformity number.
Fig. 13 is a graph showing that an intervention with sodium monosialotetrahexosyl ganglioside (GM 1) can protect against cisplatin-induced hearing impairment. FIG. 13A is a diagram of mouse cochlear Corti immunofluorescence, wherein Myo7a (green) labeled hair cells, DAPI (blue) labeled nuclei, 3mg/kg of Cisplatin (Cisplatin) injected intraperitoneally caused reduction of hair cells, and 20mg/kg of GM1 intraperitoneally prevented reduction of hair cells caused by Cisplatin (Scale Bar:50 μm). Fig. 13B is a graph of statistics of the number of hair cells with Myo7a positive signal in a 200 μm region (white dashed region) of cochlea from different groups, where P <0.001 and P <0.0001. Figures 13C-F show ABR waveforms for mice under 24KHz stimulation between different groups. Fig. 13G separately counts ABR response thresholds between different groups, where P <0.0001. Fig. 13H separately counts ABR latency at 80dB stimulation for different groups, where P <0.0001.
Fig. 14 is a graph showing that sodium monosialotetrahexosyl ganglioside (GM 1) intervention can protect against oxaliplatin-induced hearing impairment. FIG. 14A is a diagram of mouse cochlear Corti immunofluorescence, wherein Myo7a (green) labeled hair cells, DAPI (blue) labeled nuclei, and intraperitoneal injection of oxaliplatin (Oxa) 5mg/kg caused hair cell reduction, and intraperitoneal injection of GM1 20mg/kg prevented Oxa-induced hair cell reduction. Fig. 14B counts the number of Myo7a positive signal hair cells in 200 μm regions (white dashed regions) of cochlea of different groups of mice, where P <0.0001. FIGS. 14C-F show waveforms of ABR of mice under 24KHz stimulation between different groups. Fig. 14G separately counts ABR response thresholds between different groups, where P <0.0001. Fig. 14H separately counts ABR latency at 80dB stimulation for different groups, where P <0.0001.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below, unless otherwise specified, are all commercially available products.
EXAMPLE 1 action of GM1 on prevention/amelioration of cisplatin-induced mouse cochlear hair cell/hearing impairment
1. GM1 pre-protects cisplatin against damage to mouse cochlear hair cells (HEI-OC 1)
The HEI-OC1 cell experiment procedure, as shown in FIG. 1A, includes the following steps:
1. 10% complete medium configuration ratio: DMEM (45 ml) +FBS (5 ml)
Cell resuscitation: and (3) placing the frozen cells in a water bath at 33 ℃ to quickly dissolve the frozen cells, adding the dissolved cells into a preheated culture medium, placing the culture medium into an incubator, and performing full liquid exchange after 10 hours.
Cell passage: when the cell density was 80% of the bottom area of the dish, the passage of cells was performed, as observed under a microscope. Medium, PBS (cell culture grade), 0.25% Trypsin-EDTA (gibco) were pre-warmed for 15min.
(1) Cells were removed from the incubator, the supernatant was discarded, washed once with PBS, and note: when the liquid is added, the liquid is added along the wall of the dish, so that the cells are prevented from being blown up;
(2) Removing PBS, adding 1-2 ml 0.25% Trypsin-EDTA (the amount of pancreatin depends on the bottom area of the dish, and can be covered on the bottom of the dish), and digesting for 90s at 37 ℃ to see that the cells form a whole film on the bottom of the dish;
(3) Adding fresh culture medium to stop digestion, flushing down cells, transferring the cells to a centrifuge tube, and adding the cells into the centrifuge tube at 1500rpm/5min;
(4) The supernatant was discarded and the cells were resuspended by adding 1ml of medium. Cell count at a density of 1X 10 4 Passaging at each/ml.
2. Cell seed plates: the pre-digestion, resuspension and counting of the plates are the same as those of the passages
(1) The plates had two different cell densities, a high density of 3X 10 4 Individual/ml, low density 1X 10 4 Individual/ml;
(2) 100 μl of cell suspension is added to each well of a 96-well plate, and the surrounding wells are sealed by sterile water;
(3) The 96-well plate of the seeded cells was returned to the incubator for culturing.
3. Cisplatin IC 50 Measurement
(1) After the cell is seeded, placing the cell into an incubator for culturing for 24 hours, discarding the old culture medium, adding a fresh culture medium containing cisplatin with different concentrations, and continuing culturing for 24 hours or 48 hours;
(2) After the culture is finished, 10 μl of CCK8 is added into each well, the culture is continued for 4 hours, and the absorbance OD value is measured by an enzyme-labeling instrument;
(3) Calculation using graphpad according to OD valueCompound IC 50 Value according to IC 50 And selecting the concentrations of the corresponding high-damage group, medium-damage group and low-damage group to obtain the HEI-OC1 cell model with low damage, medium damage and high damage caused by cisplatin for subsequent experimental study.
4. Pre-protection of HEL-OC1 cells by GM1
(1) Cell seed plates are placed in an incubator for culturing for 24 hours;
(2) Removing the old culture medium, adding GM1 (0.01-1 mM, aqueous solution) and treating for 24 hours or 48 hours;
(3) After GM1 treatment for 24 or 48 hours, a medium containing GM1 was aspirated for configuring cisplatin;
(4) Placing a culture solution containing GM1 and cisplatin into an incubator;
(5) The treatment was continued in the incubator for 24 hours or 48 hours, then 10. Mu.l of CCK8 was added to each well, and after culturing for 4 hours, OD value was measured to calculate cell viability.
The results of the studies are shown in FIG. 1, panel B, panel C and panel D, and the results show that GM1 has a pre-protective effect in the concentration range of 0.01-1 mM.
2. GM1 pre-protects cisplatin from damage to zebra fish hair cells at 24hpf treatment
Collecting Tg (Brn 3c: EGFP) transgenic zebra fish embryos of fluorescent protein marked hair cells, incubating in a 28.5 ℃ incubator, placing 6hpf embryos with good selection status in a 96-well plate, and randomly grouping the embryos: control, GM1, cisplatin (cispratin), gm1+cisplatin, as shown in fig. 2A.
Embryos were incubated with embryo culture medium containing a concentration of GM1 (0.1 mg/ml-2 mg/ml) starting at the 8-24 hpf stage, and after 6h of GM1 treatment, a treatment with cisplatin (0.01-0.5 mg/ml) at a concentration according to the groupings was performed as shown in FIG. 2A.
The drug treatment process is carried out in an incubator at 28.5 ℃, and after the embryo grows to 72hpf, the living imaging is carried out on the juvenile zebra fish according to the required time period. The method comprises the steps of fixing young transgenic zebra fish with hair cells marked by fluorescence in 0.8% LMP agarose gel containing 0.03% tricaine anesthetic, placing the young transgenic zebra fish in a laser confocal microscope for living body three-dimensional imaging, and analyzing morphological and quantitative indexes of the hair cells.
As shown in fig. 2B and 2C, after cisplatin treatment, the arrangement of hair cells was disturbed, the hair cell body was damaged, cilia was shortened or disappeared, and the number of hair cells was significantly reduced. After GM1 pretreatment, cisplatin is added for treatment, and compared with the cisplatin treatment, the number of hair cells is obviously increased, the hair cells are arranged in a more compact cluster shape, and the cilia structure is improved.
3. GM1 pre-protects cisplatin from damage to zebra fish hair cells at 8hpf treatment
Collecting Tg (Brn 3c: EGFP) transgenic zebra fish embryos of fluorescent protein marked hair cells, incubating in a 28.5 ℃ incubator, placing 6hpf embryos with good selection status in a 96-well plate, and randomly grouping the embryos: control, GM1, cisplatin (cispratin), gm1+cisplatin, as shown in fig. 3A.
Embryos were incubated with embryo culture medium containing a concentration of GM1 (0.1 mg/ml-2 mg/ml) starting at the 8-24 hpf stage, and after 6h of GM1 treatment, a treatment with cisplatin (0.01-0.5 mg/ml) at a concentration according to the groupings was performed as shown in FIG. 3A.
The drug treatment process is carried out in an incubator at 28.5 ℃, and after the embryo grows to 72hpf, the living imaging is carried out on the juvenile zebra fish according to the required time period. The method comprises the steps of fixing young transgenic zebra fish with hair cells marked by fluorescence in 0.8% LMP agarose gel containing 0.03% tricaine anesthetic, placing the young transgenic zebra fish in a laser confocal microscope for living body three-dimensional imaging, and analyzing morphological and quantitative indexes of the hair cells.
As shown in fig. 3B and 3C, after cisplatin treatment, the arrangement of hair cells was disturbed, the hair cell body was damaged, cilia was shortened or disappeared, and the number of hair cells was significantly reduced. After GM1 pretreatment, cisplatin is added for treatment, and compared with the cisplatin treatment, the number of hair cells is obviously increased, the hair cells are arranged in a more compact cluster shape, and the cilia structure is improved.
Example 2 action of GM1 on prevention/amelioration of oxaliplatin-induced cochlear hair cell/hearing impairment in mice and zebra fish
1. GM1 pre-protects oxaliplatin against damage to mouse cochlear hair cells (HEI-OC 1)
The HEI-OC1 cell assay procedure, as shown in FIG. 4, panel A, includes the following steps:
1. 10% complete medium configuration ratio: DMEM (45 ml) +FBS (5 ml)
Cell resuscitation: and (3) placing the frozen cells in a water bath at 33 ℃ to quickly dissolve the frozen cells, adding the dissolved cells into a preheated culture medium, placing the culture medium into an incubator, and performing full liquid exchange after 10 hours.
Cell passage: when the cell density was 80% of the bottom area of the dish, the passage of cells was performed, as observed under a microscope. Medium, PBS (cell culture grade), 0.25% Trypsin-EDTA (gibco) were pre-warmed for 15min.
(1) Cells were removed from the incubator, the supernatant was discarded, washed once with PBS, and note: when the liquid is added, the liquid is added along the wall of the dish, so that the cells are prevented from being blown up;
(2) Removing PBS, adding 1-2 ml 0.25% Trypsin-EDTA (the amount of pancreatin depends on the bottom area of the dish, and can be covered on the bottom of the dish), and digesting for 90s at 37 ℃ to see that the cells form a whole film on the bottom of the dish;
(3) Adding fresh culture medium to stop digestion, flushing down cells, transferring the cells to a centrifuge tube, and adding the cells into the centrifuge tube at 1500rpm/5min;
(4) The supernatant was discarded and the cells were resuspended by adding 1ml of medium. Cell count at a density of 1X 10 4 Passaging at each/ml.
2. Cell seed plates: the pre-digestion, resuspension and counting of the plates are the same as those of the passages
(1) The plates had two different cell densities, a high density of 3X 10 4 Individual/ml, low density 1X 10 4 Individual/ml;
(2) 100 μl of cell suspension is added to each well of a 96-well plate, and the surrounding wells are sealed by sterile water;
(3) The 96-well plate of the seeded cells was returned to the incubator for culturing.
3. Compound oxaliplatin IC 50 Measurement
(1) After cell seed plates are put into an incubator for culturing for 24 hours, old culture medium is discarded, fresh culture medium containing oxaliplatin with different concentrations is added, and the culture is continued for 24 hours or 48 hours;
(2) After the culture is finished, 10 μl of CCK8 is added into each well, the culture is continued for 4 hours, and the absorbance OD value is measured by an enzyme-labeling instrument;
(3) Calculation of Compound IC using graphpad according to OD value 50 Value according to IC 50 And selecting the concentrations of the corresponding high-damage group, medium-damage group and low-damage group to obtain the low-damage, medium-damage and high-damage HEI-OC1 cell model caused by oxaliplatin for subsequent experimental study.
4. Pre-protection of HEL-OC1 cells by GM1
(1) Cell seed plates are placed in an incubator for culturing for 24 hours;
(2) Removing the old culture medium, adding GM1 (0.01-1 mM, aqueous solution) with different concentrations, and treating for 24 hours or 48 hours;
(3) After GM1 treatment for 24 or 48 hours, medium containing GM1 was aspirated to configure oxaliplatin;
(4) Placing a culture solution containing GM1 and oxaliplatin into an incubator;
(5) The treatment was continued in the incubator for 24 hours or 48 hours, then 10. Mu.l of CCK8 was added to each well, and after culturing for 4 hours, OD value was measured to calculate cell viability.
The results of the studies are shown in FIG. 4, panel B, panel C and panel D, and the results show that GM1 has a pre-protective effect in the concentration range of 0.01 to 1 mM.
2. GM1 pre-protects oxaliplatin from damage to zebra fish hair cells at 24hpf treatment
Collecting Tg (Brn 3c: EGFP) transgenic zebra fish embryos of fluorescent protein marked hair cells, incubating in a 28.5 ℃ incubator, placing 6hpf embryos with good selection status in a 96-well plate, and randomly grouping the embryos: control group, GM1 treated group, oxaliplatin (oxaliplatin) treated group, and GM1+ oxaliplatin treated group, as shown in fig. 5A.
Embryos were incubated at 24hpf with embryo culture medium containing a concentration of GM1 (0.1 mg/ml to 2 mg/ml), and after 6h of GM1 treatment, oxaliplatin (0.1 mg/ml to 1 mg/ml) treatment at a concentration according to the groupings was performed as shown in FIG. 5A.
The drug treatment process is carried out in an incubator at 28.5 ℃, and after the embryo grows to 72hpf, the living imaging is carried out on the juvenile zebra fish according to the required time period. The method comprises the steps of fixing young transgenic zebra fish with hair cells marked by fluorescence in 0.8% LMP agarose gel containing 0.03% tricaine anesthetic, placing the young transgenic zebra fish in a laser confocal microscope for living body three-dimensional imaging, and analyzing morphological and quantitative indexes of the hair cells.
As shown in fig. 5B and 5C, after oxaliplatin treatment, the arrangement of hair cells was disturbed, the hair cell body was damaged, cilia was shortened or disappeared, and the number of hair cells was significantly reduced. After GM1 pretreatment, oxaliplatin is added for treatment, so that the number of hair cells is obviously increased, the hair cells are arranged in a more compact cluster shape, and the cilia structure is improved compared with the single oxaliplatin treatment.
3. GM1 pre-protects oxaliplatin from damage to zebra fish hair cells at 8hpf treatment
Collecting Tg (Brn 3c: EGFP) transgenic zebra fish embryos of fluorescent protein marked hair cells, incubating in a 28.5 ℃ incubator, placing 6hpf embryos with good selection status in a 96-well plate, and randomly grouping the embryos: control group, GM1 treated group, oxaliplatin (oxaliplatin) treated group, and GM1+ oxaliplatin treated group, as shown in fig. 6A.
Embryos were incubated with embryo culture medium containing a concentration of GM1 (0.1 mg/ml-2 mg/ml) at the beginning of 8hpf, and after treatment with GM1 for 6h, treatment with oxaliplatin (0.1 mg/ml-1 mg/ml) at a concentration according to the groupings was performed as shown in FIG. 6A.
The drug treatment process is carried out in an incubator at 28.5 ℃, and after the embryo grows to 72hpf, the living imaging is carried out on the juvenile zebra fish according to the required time period. The method comprises the steps of fixing young transgenic zebra fish with hair cells marked by fluorescence in 0.8% LMP agarose gel containing 0.03% tricaine anesthetic, placing the young transgenic zebra fish in a laser confocal microscope for living body three-dimensional imaging, and analyzing morphological and quantitative indexes of the hair cells.
As shown in fig. 6B and 6C, after oxaliplatin treatment, the arrangement of hair cells was disturbed, the hair cell body was damaged, cilia was shortened or disappeared, and the number of hair cells was significantly reduced. After GM1 pretreatment, oxaliplatin is added for treatment, so that the number of hair cells is obviously increased, the hair cells are arranged in a more compact cluster shape, and the cilia structure is improved compared with the single oxaliplatin treatment.
EXAMPLE 3 action of GM1 in preventing/ameliorating Taxol or its combination platinum-based drug-induced Zebra fish hair cell injury/hearing injury
1. GM1 pre-protects paclitaxel against injury to zebra fish hair cells at 24hpf treatment
Collecting Tg (Brn 3c: EGFP) transgenic zebra fish embryos of fluorescent protein marked hair cells, incubating in a 28.5 ℃ incubator, placing 6hpf embryos with good selection status in a 96-well plate, and randomly grouping the embryos: control group, GM1 treated group, paclitaxel (albumin binding group consisting of 100mg paclitaxel and 900mg human serum albumin) treated group, GM1+ paclitaxel (albumin binding) treated group. As shown in FIG. 7A, the treatment group of paclitaxel (albumin-bound) (0.01 mg/ml to 1mg/ml, embryo culture fluid) was started at 48 hpf. Embryos are incubated with embryo culture solution containing a certain concentration (0.1 mg/ml-2 mg/ml) of GM1 at the beginning of 24hpf, and after GM1 treatment for 6 hours, paclitaxel (albumin binding) treatment (0.01 mg/ml-1 mg/ml, embryo culture solution) with a certain concentration can be carried out according to the grouping.
The drug treatment process was carried out in an incubator at 28.5 ℃. After the embryo develops to 72hpf, the zebra fish juvenile fish is subjected to living imaging according to the required time period. The result of three-dimensional imaging of living bodies under a laser confocal microscope is shown in FIG. 7B by fixing young transgenic zebra fish with fluorescence labeled hair cells in 0.8% LMP agarose gel containing 0.03% tricaine anesthetic; the results of analysis of the morphological, quantitative, and distribution of hair cells are shown in FIG. 7C.
After paclitaxel (albumin binding) treatment, the arrangement of hair cells is disordered, the hair cell body is damaged, cilia are shortened or disappear, and the number of hair cells is obviously reduced. After GM1 pretreatment, paclitaxel (albumin binding) treatment is added, and compared with paclitaxel (albumin binding) treatment alone, the number of hair cells is significantly increased, the hair cells are arranged in a more compact cluster, and the cilia structure is improved.
2. GM1 pre-protects paclitaxel against injury to zebra fish hair cells at 8hpf treatment
Collecting Tg (Brn 3c: EGFP) transgenic zebra fish embryos of fluorescent protein marked hair cells, incubating in a 28.5 ℃ incubator, placing 6hpf embryos with good selection status in a 96-well plate, and randomly grouping the embryos: control group, GM1 treated group, paclitaxel (albumin binding group consisting of 100mg paclitaxel and 900mg human serum albumin) treated group, GM1+ paclitaxel (albumin binding) treated group. As shown in FIG. 8A, the treatment group of paclitaxel (albumin-bound) (0.01 mg/ml to 1mg/ml, embryo culture fluid) was started at 48 hpf. At 8hpf, embryo culture solution containing GM1 at a certain concentration (0.1 mg/ml-2 mg/ml) is used for incubating embryo, and after GM1 treatment for 6h, paclitaxel (albumin binding type) treatment (0.01 mg/ml-1 mg/ml, embryo culture solution) at a certain concentration can be performed according to the grouping.
The drug treatment process was carried out in an incubator at 28.5 ℃. After the embryo develops to 72hpf, the zebra fish juvenile fish is subjected to living imaging according to the required time period. The result of the three-dimensional imaging of living bodies of the young transgenic zebra fish with the hair cells marked by fluorescence is fixed in 0.8% LMP agarose gel containing 0.03% tricaine anesthetic and placed under a laser confocal microscope is shown in FIG. 8B; the results of analysis of the morphological, quantitative, and distribution of hair cells are shown in FIG. 8C.
After paclitaxel (albumin binding) treatment, the arrangement of hair cells is disordered, the hair cell body is damaged, cilia are shortened or disappear, and the number of hair cells is obviously reduced. After GM1 pretreatment, paclitaxel (albumin binding) treatment is added, and compared with paclitaxel (albumin binding) treatment alone, the number of hair cells is significantly increased, the hair cells are arranged in a more compact cluster, and the cilia structure is improved.
3. Injury of Magnaporthe grisea cells by GM1 in combination with pre-protecting paclitaxel at 8hpf
Collecting Tg (Brn 3c: EGFP) transgenic zebra fish embryos of fluorescent protein marked hair cells, incubating in a 28.5 ℃ incubator, placing 6hpf embryos with good selection status in a 96-well plate, and randomly grouping the embryos: control group, GM1 treatment group, paclitaxel (albumin binding group consisting of 100mg paclitaxel and 900mg human serum albumin) in combination with cisplatin treatment group, and GM1+ paclitaxel (albumin binding) in combination with cisplatin treatment group. As shown in FIG. 9A, the treatment group of paclitaxel (albumin-bound) (0.01 mg/ml to 1mg/ml, embryo culture solution) in combination with cisplatin (0.01 mg/ml to 0.5mg/ml, embryo culture solution) was started at 48 hpf. At 8hpf, embryo is incubated with embryo culture solution containing GM1 at a certain concentration (0.1 mg/ml-2 mg/ml), and after GM1 treatment for 6h, paclitaxel (albumin binding type) (0.01 mg/ml-1 mg/ml, embryo culture solution) at a certain concentration can be combined with cisplatin (0.01 mg/ml-0.5 mg/ml, embryo culture solution) according to the grouping.
The drug treatment process was carried out in an incubator at 28.5 ℃. After the embryo develops to 72hpf, the zebra fish juvenile fish is subjected to living imaging according to the required time period. The result of three-dimensional imaging of living bodies under a laser confocal microscope is shown in FIG. 9B by fixing young transgenic zebra fish with fluorescence labeled hair cells in 0.8% LMP agarose gel containing 0.03% tricaine anesthetic; the results of analysis of the morphological, quantitative, and distribution of hair cells are shown in FIG. 9C.
After paclitaxel (albumin binding) combined cisplatin treatment, the arrangement of hair cells is disordered, the hair cell body is damaged, cilia are shortened or disappear, and the number of hair cells is obviously reduced. After GM1 pretreatment, paclitaxel (albumin binding) is added to the mixture for combined cisplatin treatment, so that the number of hair cells is obviously increased, and the hair cells are arranged in a more compact cluster shape and the cilia structure is improved compared with the treatment of only paclitaxel (albumin binding) combined cisplatin.
4. Injury of Magnomonic fish hair cells by GM1 in combination with oxaliplatin pre-protection of paclitaxel at 8hpf
Collecting Tg (Brn 3c: EGFP) transgenic zebra fish embryos of fluorescent protein marked hair cells, incubating in a 28.5 ℃ incubator, placing 6hpf embryos with good selection status in a 96-well plate, and randomly grouping the embryos: control group, GM1 treatment group, paclitaxel (albumin binding group consisting of 100mg paclitaxel and 900mg human serum albumin) in combination with oxaliplatin treatment group, GM1+ paclitaxel (albumin binding) in combination with oxaliplatin treatment group. As shown in FIG. 10A, the treatment group of paclitaxel (albumin-bound) (0.01 mg/ml-1 mg/ml, embryo culture broth) in combination with oxaliplatin (0.1 mg/ml-1 mg/ml, embryo culture broth) was started at 48 hpf. At 8hpf, embryo is incubated with embryo culture solution containing GM1 at a certain concentration (0.1 mg/ml-2 mg/ml), and after GM1 treatment for 6h, paclitaxel (albumin binding type) (0.01 mg/ml-1 mg/ml, embryo culture solution) at a certain concentration can be combined with oxaliplatin (0.1 mg/ml-1 mg/ml, embryo culture solution) according to the grouping.
The drug treatment process was carried out in an incubator at 28.5 ℃. After the embryo develops to 72hpf, the zebra fish juvenile fish is subjected to living imaging according to the required time period. The result of three-dimensional imaging of living bodies under a laser confocal microscope is shown in FIG. 10B by fixing young transgenic zebra fish with fluorescence labeled hair cells in 0.8% LMP agarose gel containing 0.03% tricaine anesthetic; the results of analysis of the morphological, quantitative, and distribution of hair cells are shown in FIG. 10C.
After paclitaxel (albumin binding) combined oxaliplatin treatment, the arrangement of hair cells is disordered, the hair cell body is damaged, cilia are shortened or disappear, and the number of hair cells is obviously reduced. After GM1 pretreatment, paclitaxel (albumin binding) combined oxaliplatin is added for treatment, and compared with paclitaxel (albumin binding) combined oxaliplatin alone, the number of hair cells is significantly increased, the hair cells are in a more compact cluster arrangement, and the cilia structure is improved.
EXAMPLE 4 action of GM1 in preventing/ameliorating cisplatin, oxaliplatin and paclitaxel induced mouse hair cell injury/hearing impairment
1. GM1 prevents/ameliorates cisplatin-induced cochlear hair cell injury in mice
The newborn mice were treated with the drugs according to the experimental group, with a GM1 dose of 200mg/kg and a cisplatin dose of 5mg/kg. After the drug treatment was completed, the mice tempral bone was removed and placed on an ice box to rapidly dissect the cochlea. 10mm coverslips were applied with packed cells-tak, each of which was 0.5. Mu.l, rounded to a diameter of 5mm, and dried (freshly applied). The coated cover glass is placed in a culture dish with cochlea, the cochlea is adhered to the glass with the right side upwards, the adhered glass is placed in a 4-hole culture dish (3 ml of PBS is added before and placed on ice), and after all the glass slides are adhered, 16% PFA is added in the four-hole culture dish, so that the solution is finally 4% PFA. Fixed for 1h, washed 3 times with PBST, blocked medium for 1-2h (100 μl per well), and in PBT-1 with primary antibody added thereto overnight at 4deg.C, after 3 times with PBST, secondary antibody was added thereto at room temperature for 1h (100 μl per well). PBST was washed 3 times, 6. Mu.l DAKO was added to each sample, covered with a coverslip, and sealed with nail polish. The morphology of the cochlea hair cells of the mice was observed under a fluorescence microscope, and the results are shown in fig. 11A. Hair cell phenotype counts showed that cisplatin had significant mouse cochlear hair cytotoxicity, and GM1 pretreatment had significant protective effects, reducing hair cell loss and deformity number (fig. 11B).
2. GM1 prevents/improves mouse cochlear hair cell injury caused by oxaliplatin in combination with paclitaxel
The neonatal mice were dosed with drug treatment according to the experimental group with a dose of 200mg/kg GM1 and 5mg/kg oxaliplatin and paclitaxel. After the drug treatment was completed, the mice tempral bone was removed and placed on an ice box to rapidly dissect the cochlea. 10mm coverslips were applied with packed cells-tak, each of which was 0.5. Mu.l, rounded to a diameter of 5mm, and dried (freshly applied). The coated cover glass is placed in a culture dish with cochlea, the cochlea is adhered to the glass with the right side upwards, the adhered glass is placed in a 4-hole culture dish (3 ml of PBS is added before and placed on ice), and after all the glass slides are adhered, 16% PFA is added in the four-hole culture dish, so that the solution is finally 4% PFA. Fixed for 1h, washed 3 times with PBST, blocked medium for 1-2h (100 μl per well), and in PBT-1 with primary antibody added thereto overnight at 4deg.C, after 3 times with PBST, secondary antibody was added thereto at room temperature for 1h (100 μl per well). PBST was washed 3 times, 6. Mu.l DAKO was added to each sample, covered with a coverslip, and sealed with nail polish. The morphology of the cochlea hair cells of the mice was observed under a fluorescence microscope, and the results are shown in fig. 12A. Hair cell phenotype counts showed that cisplatin had significant mouse cochlear hair cytotoxicity, and GM1 pretreatment had significant protective effects, reducing hair cell loss and number of deformities (fig. 12B).
3. Research on prevention/improvement of mouse cochlear hair cell injury/hearing injury caused by cisplatin and oxaliplatin by GM1
Experimental materials
1. Experimental animals: c57BL/6 male mice at 8 weeks of age, supplied by the university of Nantong laboratory animal center.
2. Experimental reagent:
3. experimental equipment:
experimental method
1. Grouping and administration of laboratory animals
1) Cisplatin grouping and administration
Using a prospective random control experiment, the random number table method divided the experimental mice into the following four groups of 10 mice each: control group (Vehicle), cisplatin group (cispratin), GM1+ Cisplatin group (GM 1& cispratin), GM1 group (GM 1). Cisplatin group mice were given intraperitoneal injections of cisplatin at 3mg/kg for 7 days; GM1 group mice were given intraperitoneal injections of GM1 at 20mg/kg for 7 days; GM1+ cisplatin group mice were treated 7 days in advance, were intraperitoneally injected with GM1 20mg/kg, and on day 8, were intraperitoneally injected with cisplatin 3mg/kg for 7 days; the control group was given only an equal amount of physiological saline by intraperitoneal injection. Four groups were re-examined for auditory brainstem response (auditory brainstem response, ABR) one day after withdrawal, and mice were sacrificed after ABR examination to take cochlea for subsequent morphological and molecular biological experiments.
2) Oxaliplatin grouping and administration
Using a prospective random control experiment, the animals mice were divided into the following four groups of 8 mice each by the random digital table method: control group (Vehicle), oxaliplatin group (Oxa), GM1+ oxaliplatin group (GM 1& Oxa), GM1 group (GM 1). Oxaliplatin mice were given 5mg/kg of oxaliplatin for 7 consecutive days; GM1 group mice were given intraperitoneal injections of GM1 at 20mg/kg for 7 days; the mice in the group of GM1 plus oxaliplatin are treated for 7 days in advance, and are intraperitoneally injected with GM1 to 20mg/kg, and oxaliplatin to 5mg/kg at day 8 for 7 days; the control group was given only an equal amount of physiological saline by intraperitoneal injection. Four groups of mice were sacrificed one day after drug withdrawal and the mice were sacrificed after ABR examination for subsequent morphological and molecular biology experiments.
2. ABR test
ABR is a sound-induced neural response commonly used to evaluate auditory function in humans and experimental animals. In mice, ABR detection will appear in 5 waveforms, designated wave i, wave ii, wave iii, wave iv, and wave v, respectively, where wave i receives the neural signal from the acoustic nerve, wave ii receives the neural signal from the cochlea, wave iii receives the neural signal from the superior olive kernel, wave iv receives the neural signals from the periolive kernel and the lateral colliculus nuclei, and wave v receives the neural signal from the inferior colliculus nucleus.
ABR test was performed in a sound-proof room, before ABR test was performed, mice were weighed, 4% chloral hydrate was intraperitoneally injected for anesthetizing animals, subcutaneous needle electrodes were inserted into the top-of-cranium mesoderm (+electrode), mastoid (-electrode) and hind legs (ground) of the mice, respectively, click stimulation and short pure tone stimulation of different frequencies of 4KHz, 8KHz, 16KHz, 24KHz, 32KHz were applied through speaker monophonic channels, respectively, repeated stimulation was performed in 10dB steps starting from a sound pressure level of 90dB, and ABR waveform charts were recorded. After the experiment is finished, the response threshold and latency change of the recorded wave II are counted.
3. Mouse cochlea Corti immunofluorescence staining
After the ABR experiment was completed, 5 mice were randomly selected from each group for immunofluorescent staining. After the inner ear of the mouse was fixed with 4% pfa and decalcified with EDTA, the cochlea of the mouse was carefully peeled off under a microscope. Dissecting the mouse basement membrane in pre-chilled PBS; coating a 10mm cover glass with a cell-tak, coating into a circle with the diameter of 5mm, and airing (freshly coated); placing the coated cover glass into a culture dish with a cochlea basement membrane of a mouse, adhering the cochlea to the glass, and leading the cochlea to face upwards (with a protrusion on the front surface) when adhering; placing the adhered slide in a 4-hole culture dish (3 ml of PBS is added in advance and placed on ice), and washing with PBS three times; after one hour of blocking at room temperature, the capillary cell marker Myosin7a (Myo 7 a) primary antibody was added and incubated overnight at 4 ℃; rinsing with PBS for 3 times, and incubating the corresponding secondary antibodies for 2 hours at room temperature in proportion within 5 minutes each time; rinsing with PBS 3 times for 5 min, adding 10 μl DAPI (4', 6-diamidino-2-phenylindole, a fluorescent dye capable of binding strongly to DNA) containing caplets on each slide, covering with 10mm coverslips, and sealing with nail polish; pictures were taken using a confocal microscope.
4. Data processing
The images taken by laser confocal were all analyzed using Imaris x 64.0. A software. All data obtained from the experiments were treated with Graphpad Prism8, one-way ANOVA was used for single-factor multi-group differential comparison, two-way ANOVA was used for multi-factor comparison between Two groups, and P <0.05 indicated that the differences were statistically significant.
Experimental results and conclusions
1. GM1 prevents cisplatin-induced damage to Corti hair cells and ABR response threshold abnormalities
The Corti device is a screw located on the basal membrane of the cochlear canal, consisting of inner and outer hair cells, supporting cells and a covering membrane (tectorial membrane), which are the main components of auditory receptors. On the worm shaft side there are single rows of Inner Hair Cells (IHCs) and on the outer side there are 3 or more rows of Outer Hair Cells (OHCs). The heights of hair cells at different parts of the basement membrane are different, and the hair cells gradually increase from the bottom of the volute to the top of the volute. The static cilia of the cells at the fundus of the snail become short, the static cilia near the tip of the snail become longer gradually, and the sensitive wavelength becomes longer gradually. The gradient of the height of these cochlear hair cells and the length of the static cilia is the morphological basis for producing cochlear wavelength alignment and wave-regulating function. To investigate whether GM1 has a preventive protective effect on cisplatin-induced hair cell damage, mouse cochlear corpi hair cells were detected by immunofluorescent staining after intraperitoneal injection of cisplatin. The experimental results are shown in fig. 13 (a-B) and table 1, and the numbers of Myo7a positive signal hair cells in the top (Apex), middle (Middle) and Base (Base) of the left and right cochlea of the mice are counted, respectively, and the results show that the intraperitoneal injection of cisplatin 3mg/kg causes the reduction of the number of the cochlea hair cells of the mice compared with the control group, and GM1 can prevent the reduction of the hair cells caused by cisplatin.
Since wave ii of ABR test received nerve signals of cochlear nucleus, experimental statistics recorded the response threshold and latency change of wave ii between different groups, and experimental results are shown in fig. 13 (C-H) and tables 2 and 3, cisplatin can cause ABR response threshold and ABR response latency abnormality of mice, which is manifested by an increase in response threshold and prolongation of latency, while GM1 can prevent hearing abnormality of mice. Experimental results suggest that pretreatment with GM1 can prevent cisplatin hearing impairment.
Tables 1-3 illustrate: tables 1-3 are all data relating to 10 mice per group, with no order.
TABLE 1 GM1 prevention of cisplatin-induced hearing impairment-Corti device Myo7a Positive hair cell count
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TABLE 2 GM1 prevention of cisplatin induced hearing impairment-ABR behavioural test latency (ms)
TABLE 3 GM1 prevention of cisplatin induced hearing impairment-ABR behavioural experimental hearing threshold (dB)
4. GM1 prevents oxaliplatin-induced hearing impairment-Corti hair cell injury and ABR response threshold abnormality
After 5mg/kg of oxaliplatin was intraperitoneally injected, the mouse cochlear Corti hair cells were examined by immunofluorescence staining, and the experimental results are shown in FIG. 14 (A-B) and Table 4, in which oxaliplatin caused a decrease in the number of mouse cochlear hair cells, while GM1 prevented the decrease in hair cells caused by oxaliplatin. The results of the experiments are shown in FIG. 14 (C-H) and tables 5 and 6, and oxaliplatin can cause abnormality of the ABR response threshold and the ABR response latency of mice, which is manifested by an increase in the response threshold and an increase in the latency, while GM1 can prevent abnormal hearing behavior of mice. Experimental results suggest that pretreatment with GM1 can prevent oxaliplatin hearing impairment.
Tables 4-6 illustrate: table 4 shows the relevant data of 10 mice per group, without sequence. Tables 5 and 6 show the relevant data of 8 mice in each group, without any sequence.
TABLE 4 GM1 prevention of oxaliplatin induced hearing impairment-Corti device Myo7a positive hair cell count
TABLE 5 GM1 prevention of oxaliplatin induced hearing impairment-ABR behavioral experimental latency (ms)
TABLE 6 GM1 prevention of oxaliplatin induced hearing impairment-ABR behavioural experimental hearing threshold (dB)
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (7)
1. Use of ganglioside GM1 in the manufacture of a medicament for preventing, alleviating or treating hearing impairment caused by a chemotherapeutic agent selected from cisplatin, oxaliplatin, paclitaxel, a combination of cisplatin and paclitaxel, a combination of oxaliplatin and paclitaxel, said hearing impairment comprising hair cell impairment of a patient of varying degrees comprising: the disappearance/decrease of normal cells, the increase of abnormal cells, the abnormality of cell location or cell relative location, the abnormality of characteristic index molecules in cells.
2. The use of claim 1, wherein the patient comprises a tumor patient selected from patients with multiple myeloma, digestive tract tumor, respiratory tract tumor, head and neck tumor, breast cancer, and reproductive system tumor.
3. The use according to claim 1, wherein the ganglioside GM1 is sodium monosialotetrahexosyl ganglioside.
4. The use according to claim 1, wherein the ganglioside GM1 is in a formulation form selected from the group consisting of injectable, oral, topical or topical formulations.
5. The use according to claim 1, wherein the ganglioside GM1 is in the form of an injectable formulation.
6. The use of claim 1, wherein the ganglioside GM1 is administered before, during or after the administration of the chemotherapeutic agent.
7. Use of ganglioside GM1 in the manufacture of a medicament for use in combination with a chemotherapeutic agent for preventing hearing impairment in a patient with a tumor, wherein the chemotherapeutic agent is selected from cisplatin, oxaliplatin, paclitaxel, a combination of cisplatin and paclitaxel, or a combination of oxaliplatin and paclitaxel, the hearing impairment comprising hair cell impairment of varying degrees, comprising: the disappearance/decrease of normal cells, the increase of abnormal cells, the abnormality of cell location or cell relative location, the abnormality of characteristic index molecules in cells.
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| Cisplatin and oxaliplatin are toxic to cochlear outer hair cells and both target thioredoxin reductase in organ of Corti cultures;PASCAL DAMMEYER 等;Acta Oto-Laryngologica.(第134期);448–454 * |
| Ototoxicity of paclitaxel in rat cochlear organotypic cultures;Yang Dong 等;Toxicology and Applied Pharmacology(第280期);526-533 * |
| The Ganglioside Monosialotetrahexosylganglioside Protects Auditory Hair Cells Against Neomycin-Induced Cytotoxicity Through Mitochondrial Antioxidation: An in vitro Study;Yujin Li 等;Frontiers in Cellular Neuroscience;doi: 10.3389/fncel.2021.751867 * |
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