Application of maslinic acid as cis-platinum toxicity protective agent

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to application of maslinic acid as a cis-platinum toxicity protective agent. The maslinic acid disclosed by the invention can be used for preventing ototoxicity induced by cis-platinum by recovering the activity of mouse cochlear hair cells SLC7A11, and can be used for repairing mouse hearing impairment induced by cis-platinum. Experimental results show that maslinic acid can recover activity reduction of SLC7A11 caused by cis-platinum, so that the level of LPO in cells is reduced, the survival rate of HEI-OC1 cells is increased, and the anti-cancer effect of cis-platinum is not affected; animal experiments also show that maslinic acid can inhibit mouse hearing impairment induced by cis-platinum. Therefore, maslinic acid can reduce the ear toxicity of cis-platinum and exert an inner ear protection effect.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and particularly relates to application of maslinic acid as a protective agent against cisplatin ototoxicity. Background Art

[0002] Platinum drugs are a common anti-tumor drug. Due to their broad spectrum and high efficacy, they are used to treat a variety of human cancers, including bladder cancer, head and neck cancer, lung cancer, ovarian cancer, and testicular cancer. Among the many chemotherapy drugs widely used in cancer, cisplatin is the most eye-catching one and the first platinum compound approved by the FDA for cancer treatment. During tumor treatment, repeated intravenous administration of cisplatin can cause it to accumulate in specific areas of the body, causing toxicity to the human body, including nephrotoxicity, ototoxicity, hepatotoxicity, peripheral neuropathy, bone marrow suppression, and retinopathy. Among all these toxic side effects, sensorineural hearing loss caused by cisplatin is irreversible and therefore receives the greatest attention.

[0003] Maslinic acid (MA) is a pentacyclic triterpenoid compound found primarily in natural plants such as olive and hawthorn, and is also abundant in traditional herbs such as Platycodon grandiflorum and Patchouli. Maslinic acid is primarily extracted from olive, but can also be prepared semi-synthetically using oleanolic acid. Maslinic acid exhibits anti-inflammatory, antioxidant, anti-tumor, hypoglycemic, and neuroprotective effects. This study demonstrates for the first time that maslinic acid protects against cisplatin-induced cochlear hair cell death in mice.

[0004] Literature reports indicate that cisplatin can induce ferroptosis in cochlear hair cells, leading to hearing loss. The present invention found that maslinic acid can protect against cisplatin-induced cochlear hair cell death and, by restoring SLC7A11 activity, reduce intracellular lipid peroxidation levels, protect mouse cochlear hair cells from damage without affecting the anti-cancer effect of cisplatin. In vivo experiments also showed that maslinic acid can inhibit cisplatin-induced hearing loss. Therefore, maslinic acid has the potential to be developed as a protective agent for cisplatin-induced hearing loss. Currently, there are no reports on the use of maslinic acid to intervene in cisplatin-induced hearing loss. In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention overcomes the deficiencies in the prior art and provides the following technical solutions:

[0006] The first aspect of the present invention is to provide the use of Maslinic acid (MA) and its derivatives in the preparation of ototoxic protective agents.

[0007] Furthermore, the ototoxicity is caused by tumor chemotherapy drugs.

[0008] Furthermore, the tumor chemotherapy drug is cisplatin.

[0009] Furthermore, the maslinic acid derivative is a pharmaceutically acceptable salt thereof.

[0010] Furthermore, the structural formula of the maslinic acid is:

[0011]

[0012] Furthermore, the dosage form of the protective agent includes at least one of a suspension, granules, capsules, powders, tablets, pills, injections, suppositories, and drops.

[0013] Furthermore, the administration route of the protective agent includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

[0014] Furthermore, the dosage of the protective agent is 100-200 mg / kg / Day calculated as the effective amount of maslinic acid per kilogram of body weight per day, and the administration period is 5-10 days; preferably, the protective agent is administered by gavage, and the dosage is 150 mg / kg / Day.

[0015] The second aspect of the present invention is to provide the use of maslinic acid in preparing a preparation for preventing and treating the decrease of reduced glutathione (GSH) in the body.

[0016] Furthermore, the decrease in GSH is caused by tumor chemotherapy drugs.

[0017] Furthermore, the tumor chemotherapy drug is cisplatin.

[0018] Furthermore, the maslinic acid derivative is a pharmaceutically acceptable salt thereof.

[0019] Furthermore, the structural formula of the maslinic acid is:

[0020]

[0021] Furthermore, the dosage form of the preparation includes at least one of a suspension, granules, capsules, powders, tablets, pills, injections, suppositories, and drops.

[0022] Furthermore, the administration route of the preparation includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

[0023] Furthermore, the dosage of the preparation is 100-200 mg / kg / Day, calculated as the effective amount of maslinic acid per kilogram of body weight per day, and the administration period is 5-10 days; preferably, the protective agent is administered by gavage, and the dosage is 150 mg / kg / Day.

[0024] The third aspect of the present invention is to provide the use of maslinic acid in preparing a preparation for reversing the decreased activity of SLC7A11 in cochlear hair cells.

[0025] Furthermore, the decrease in the activity of SLC7A11 in the cochlear hair cells is caused by tumor chemotherapy drugs.

[0026] Furthermore, the tumor chemotherapy drug is cisplatin.

[0027] Furthermore, the maslinic acid derivative is a pharmaceutically acceptable salt thereof.

[0028] Furthermore, the structural formula of the maslinic acid is:

[0029]

[0030] Furthermore, the dosage form of the preparation includes at least one of a suspension, granules, capsules, powders, tablets, pills, injections, suppositories, and drops.

[0031] Furthermore, the administration route of the preparation includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

[0032] Furthermore, the dosage of the preparation is 100-200 mg / kg / Day, calculated as the effective amount of maslinic acid per kilogram of body weight per day, and the administration period is 5-10 days; preferably, the protective agent is administered by gavage, and the dosage is 150 mg / kg / Day.

[0033] The fourth aspect of the present invention is to provide the use of maslinic acid in preparing a preparation of a cochlear hair cell protective agent, wherein the use is in vitro.

[0034] Furthermore, the protection is to increase the lipid peroxidation level of cochlear hair cells.

[0035] Furthermore, the protection is to inhibit the death of mouse cochlear hair cells induced by the SLC7A11 inhibitor sulfasalazine (SAS).

[0036] Furthermore, the protection is the reversal of cisplatin-induced cochlear hair cell damage.

[0037] The beneficial effects of the present invention include:

[0038] 1. The present invention discovered for the first time that maslinic acid can protect mice from hearing loss induced by cisplatin.

[0039] 2. The present invention discovered for the first time that maslinic acid can inhibit cisplatin-induced death of cochlear hair cells in HEI-OC1 mice and reverse the increase in lipid peroxidation levels in cochlear hair cells in mice induced by cisplatin.

[0040] 3. The present invention discovered for the first time a new mechanism by which maslinic acid protects against cisplatin-induced ototoxicity, namely, it exerts its protective effect by restoring the activity of SLC7A11. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 MA can restore cisplatin (CP)-induced hearing loss in mice;

[0042] Note: (A) Changes in click ABR hearing thresholds of mice in the control group (Con), drug-treated group (high MA), model group (M), and model-treated group (M + high MA). (B, C, D) Changes in hearing thresholds at different frequencies (8 kHz, 16 kHz, and 24 kHz) of mice in the control group (Con), drug-treated group (high MA), model group (M), and model-treated group (M + high MA). Data are expressed as mean ± standard deviation. Compared with the control group, **P < 0.01, ***P < 0.001; compared with the model-treated group, **P < 0.01, ***P < 0.001.

[0043] Figure 2 MA can restore the decrease of GSH (reduced glutathione) in mice caused by cisplatin (CP);

[0044] Note: Changes in serum GSH levels in mice in the control group (Con), drug-treated group (high MA), model group (M), and model-treated group (M+high MA). Compared with the control group, **P<0.01, ***P<0.001; compared with the cisplatin-treated group, **P<0.01, ***P<0.001.

[0045] Figure 3 MA can inhibit cisplatin (CP)-induced HEI-OC1 cell death;

[0046] Notes: (A) Cell viability of HEI-OC1 cells after 24-h treatment with CP (20 μM) alone or in combination with different concentrations of MA; (B) Crystal violet staining of HEI-OC1 cells after 24-h treatment with CP (20 μM), MA (30 μM), or the combination of CP and MA. Data are expressed as mean ± SD, n = 3. **P < 0.01, ***P < 0.001 compared with the control group; **P < 0.01, ***P < 0.001 compared with the cisplatin-only group.

[0047] Figure 4 MA can restore the inhibition of SLC7A11 activity induced by cisplatin (CP);

[0048] Note: (A-B) Cystine uptake capacity and intracellular cysteine ​​levels in HHEI-OC1 cells treated with CP (20 μM), MA (30 μM) alone, or CP and MA co-treatment for 17 hours. Data are expressed as mean ± SD, n = 3. *P < 0.05, **P < 0.01 compared with the control group; **P < 0.01, ***P < 0.001 compared with the CP alone group.

[0049] Figure 5 MA can alleviate the elevated lipid peroxidation (LPO) level in HEI-OC1 cells induced by cisplatin (CP);

[0050] Note: LPO levels in HEI-OC1 cells were measured by flow cytometry after 10 hours of treatment with MA (30 μM), CP (20 μM), or both. Data are expressed as mean ± SD, n = 3. **P < 0.01, ***P < 0.001 compared with the control group; **P < 0.01, ***P < 0.001 compared with the cisplatin-only group.

[0051] Figure 6 MA can inhibit sulfasalazine (SAS)-induced HEI-OC1 cell death;

[0052] Notes: (A) Cell viability of HEI-OC1 cells after 24 h of treatment with SAS (300 μM) alone or in combination with various concentrations of MA. (B) Effects of SAS (300 μM) alone or in combination with various concentrations of MA (10 μM, 20 μM, and 30 μM) on HEI-OC1 cell morphology. Data are expressed as mean ± standard deviation (SD), n = 3. Compared with the control group, **P < 0.01, ***P < 0.001; compared with the cisplatin-only group, **P < 0.01, ***P < 0.001.

[0053] Figure 7 MA can alleviate the elevated lipid peroxidation (LPO) level in HEI-OC1 cells induced by SAS;

[0054] Note: LPO levels in HEI-OC1 cells were measured by flow cytometry after 10 hours of treatment with MA (30 μM), SAS (300 μM), or both. Data are expressed as mean ± SD, n = 3. Compared with the control group, **P < 0.01, ***P < 0.001; compared with the cisplatin-only group, **P < 0.01, ***P < 0.001.

[0055] Figure 8 MA had no effect on the anticancer activity of cisplatin (CP) against A549 cells;

[0056] Note: Cell viability of A549 cells treated with CP (20 μM) alone or with MA (30 μM) for 24 hours. Data are expressed as mean ± SD, n = 3. **P < 0.01 compared with the control group; ns indicates no statistically significant difference compared with the CP-only treatment group. DETAILED DESCRIPTION

[0057] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods described are all conventional methods unless otherwise specified. The materials described can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute undue limitations of the present invention. It should be noted that, unless there is a conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0058] The term "SLC7A11" stands for solute carrier family 7 member 11 (SLC7A11). SLC7A11 is located on the cell membrane and transports cystine into the cell at a 1:1 ratio while exporting glutamate to the extracellular space. After entering the cell, cystine is reduced to cysteine ​​by NADPH. Under the enzymatic action of NADPH, cysteine ​​synthesizes GSH with glycine and glutamate. GSH can reduce lipid peroxides to non-toxic alcohols under the action of GPX4, thereby inhibiting the occurrence of ferroptosis.

[0059] The term "SAS" stands for sulfasalazine, an azo compound of salicylic acid and sulfapyridine. SAS inhibits SLC7A11, a key antioxidant in the ferroptosis regulatory network, leading to GSH depletion and inhibition of GPX4 activity, thus defeating the ferroptosis defense system within the cell and causing ferroptosis.

[0060] Example 1 Effect of MA on Cisplatin-Induced Hearing Loss in Mice

[0061] Experimental animal groups: 40 male C57BL / 6J mice, 6-8 weeks old, were randomly divided into 5 groups, 8 mice in each group, namely control group (Con), drug group (MA), model group (CP), and model drug group (CP+MA).

[0062] Hearing loss model: A hearing loss model was established in mice by intraperitoneal injection of cisplatin. The model group and the model treatment group were intraperitoneally injected with 5 mg / kg of cisplatin (CP) for 4 consecutive days, while the control group and the treatment group were intraperitoneally injected with the corresponding volume of normal saline.

[0063] Experimental Methods: After adaptive culture, the MA and model groups (CP+high MA) were gavaged with 150 mg / kg maslinic acid for eight consecutive days, while the control group was gavaged with the solvent (5% DMSO + 95% saline) for eight consecutive days. Nine days after adaptive culture, modeling began. The model group (CP) was intraperitoneally injected with 5 mg / kg cisplatin for four consecutive days; the model group (CP+MA) was gavaged with maslinic acid first, followed by an intraperitoneal injection of cisplatin two hours later; and the control group was intraperitoneally injected with saline for four consecutive days.

[0064] After modeling, auditory brainstem response (ABR) testing was performed using a TDT (RZ6) device to measure hearing ability, and changes in hearing thresholds were compared between groups. Following the hearing test, whole blood was collected from the mice via eyeball sampling. After the blood sedimented and separated, it was centrifuged and serum was collected. A micro-reduced glutathione (GSH) assay kit was then used to measure GSH levels in the mouse serum.

[0065] like Figure 1 As shown in (A), the hearing threshold of the model group (CP) increased by 70% compared with the control group (Con); the hearing threshold of the model administration group (CP+MA) decreased by 37.5% compared with the model group (CP).

[0066] like Figure 1 As shown in (B), at a frequency of 8 kHz, the hearing threshold of the model group (CP) increased by 62% compared with the control group (Con); the hearing threshold of the model drug administration group (CP+MA) decreased by 31.5% compared with the model group (CP). Figure 1 As shown in (C), at a frequency of 16KHz, the hearing threshold of the model group (CP) increased by 69.7% compared with the control group (Con); the hearing threshold of the model drug administration group (CP+MA) decreased by 46% compared with the model group (CP). Figure 1 As shown in (D), at a frequency of 24 kHz, the hearing threshold of the model group (CP) increased by 69.7% compared with the control group (Con); the hearing threshold of the model medication group (CP+MA) decreased by 46% compared with the model group (CP).

[0067] like Figure 2As shown in the results, the GSH content in the model group (CP) decreased by 78.8% compared with the control group (Con); the GSH content in the model administration group (CP+MA) increased by 25.8% compared with the model group (CP). Conclusion: MA can restore CP-induced hearing loss in mice.

[0068] Effects of 2MA and CP on the viability of HEI-OC1 cells

[0069] Experimental Methods: HEI-OC1 cells were cultured in DMEM supplemented with 10% FBS in a 5% CO2 atmosphere at 37°C. When the cells reached 80-90% confluency, they were passaged or split accordingly. 24 hours after splitting, when the cells reached approximately 30-40% confluency, HT22 cells were treated with CP (20 μM) alone or in combination with MA (30 μM) for 48 hours. Cell viability was determined by crystal violet staining.

[0070] like Figure 3 As shown in Figures AB, crystal violet staining showed significant differences between CP treatment alone and combined treatment with CP and MA. Treatment of HEI-OC1 cells with 20 μM CP for 48 h decreased the cell viability by 55.47% compared to the control group, whereas combined treatment with 30 μM MA and CP increased the cell viability by 19.00% compared to the CP treatment alone group.

[0071] Conclusion: MA can inhibit CP-induced HEI-OC1 cell death.

[0072] Example 3 Effects of MA and CP on SLC7A11 activity in HEI-OC1 cells

[0073] Experimental method: According to the kit instructions, the cystine uptake capacity and intracellular cysteine ​​content of HT22 cells were measured after 17 hours of treatment with MA (30 μM), CP (20 μM) alone or both.

[0074] Depend on Figure 4 As shown in Figure AB, CP treatment significantly reduced the cystine uptake capacity and intracellular cysteine ​​content of HEI-OC1 cells, while MA treatment inhibited the above changes, improved the cystine uptake capacity of HEI-OC1 cells, and increased the intracellular cysteine ​​content.

[0075] Conclusion: MA can protect the cochlear hair cells from damage by restoring the CP-induced inhibition of SLC7A11 activity and reducing the level of LPO.

[0076] Example 4 Effects of MA and CP on LPO levels in HEI-OC1 cells

[0077] Experimental method: Liperfluo fluorescence probe method was used to determine the LPO level of HEI-OC1 cells 12 hours after treatment with CP (20μM), MA (30μM) alone or both.

[0078] like Figure 5 As shown in Figures AB, LPO levels increased by 21.89% in the CP-treated group compared to the control group, whereas MA-treated cells decreased LPO levels by approximately 24.14% compared to the CP-treated group. MA significantly inhibited the CP-induced increase in LPO levels in HEI-OC1 cells, reduced lipid peroxidation, and thereby enhanced the cells' antioxidant capacity, ultimately protecting them from CP-induced cell death. Conclusion: MA can alleviate the CP-induced increase in lipid peroxidation in HEI-OC1 cells.

[0079] Example 5 Effects of MA and SAS on the viability and morphology of HEI-OC1 cells

[0080] Experimental Methods: HEI-OC1 cells were cultured in DMEM supplemented with 10% FBS in a 5% CO2 atmosphere at 37°C. When the cells reached 80-90% confluency, they were passaged or split accordingly. 24 hours after splitting, when the cells reached approximately 30-40% confluency, HT22 cells were treated with SAS (300 μM) alone or in combination with various concentrations of MA (10, 20, and 30 μM) for 24 hours. Cell viability was determined by crystal violet staining.

[0081] like Figure 6 As shown in Figures AB, in the control group, HEI-OC1 cells adhered firmly to the wall, exhibited a spindle-shaped structure, and showed tight cell connections. However, after 24 hours of SAS treatment, many cells lost their normal morphology, becoming wrinkled, rounded, and even appearing as black dots. The cell density was significantly reduced, and floating dead cells were observed. Compared with the SAS group, the cell density and morphology of the group co-treated with SAS and MA were significantly improved, indicating that MA has a significant protective effect against SAS-induced damage in HEI-OC1 cells. Crystal violet staining revealed that HT22 cells treated with 300 μM SAS for 24 hours decreased their cell viability by 63.04% compared with the control group. However, co-treatment with 10, 20, and 30 μM MA and SAS increased their cell viability by 12.77%, 28.9%, and 59.79%, respectively, compared with SAS alone. MA alone had no significant effect on HEI-OC1 cell viability.

[0082] Conclusion: MA can inhibit SAS-induced HEI-OC1 cell death.

[0083] Example 6 Effects of MA and SAS on LPO levels in HEI-OC1 cells

[0084] Experimental method: Liperfluo fluorescence probe method was used to determine the LPO level of HEI-OC1 cells 12 hours after treatment with SAS (300μM), MA (30μM) alone or both.

[0085] like Figure 7 As shown in AB, the LPO level in the SAS alone treatment group increased by 38.79% compared with the control group, while the intracellular LPO level after MA treatment decreased by about 42.94% compared with the SAS alone treatment group. MA significantly inhibited the SAS-induced increase in LPO levels in HEI-OC1 cells, reduced the level of lipid peroxidation, and thereby increased the antioxidant capacity of the cells, ultimately protecting the cells from SAS-induced death.

[0086] Conclusion: MA can alleviate the elevated lipid peroxidation level in HEI-OC1 cells induced by SAS.

[0087] Case Study 7: Effect of MA on the Anticancer Effect of CP

[0088] Experimental method: A549 cells were treated with CP (20 μM) alone or in combination with MA (30 μM) for 24 h, and the cell viability was determined by crystal violet staining.

[0089] The results are as follows Figure 8 As shown in AB, the cell survival rate of A549 cells treated with 30 μM CP for 24 h decreased by 45.11% compared with the control group, while MA treatment had no effect on CP-induced A549 cell death.

[0090] Conclusion: MA has no effect on the anticancer effect of CP.

[0091] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. Use of maslinic acid and its derivatives in the preparation of ototoxic protective agents, characterized in that: The ototoxicity is caused by tumor chemotherapy drugs; the tumor chemotherapy drugs are cisplatin.

2. The application according to claim 1, characterized in that The maslinic acid derivative is a pharmaceutically acceptable salt thereof.

3. The use according to claim 1 or 2, characterized in that The structural formula of the maslinic acid is:

4. The use according to claim 1 or 2, characterized in that The dosage form of the protective agent includes at least one of suspension, granules, capsules, powders, tablets, pills, injections, suppositories, and drops; the administration route of the protective agent includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

5. The use according to claim 4, characterized in that The dosage of the protective agent is 100-200 mg / kg / Day calculated as the effective amount of maslinic acid per kilogram of body weight per day, and the administration time is 5-10 days.

6. Use of maslinic acid in preparing a preparation of a cochlear hair cell protective agent, characterized in that: The application is an in vitro application, characterized in that the protection is to increase the lipid peroxidation level of cochlear hair cells.

7. The use according to claim 6, characterized in that The protection is to inhibit the death of mouse cochlear hair cells induced by SLC7A11 inhibitor sulfasalazine (SAS).

8. The use according to claim 6, characterized in that The protection is to reverse the damage of cochlear hair cells induced by cisplatin.

9. Use of maslinic acid in preparing a preparation for preventing and treating the reduction of reduced glutathione (GSH) in the body; characterized in that, The GSH reduction is caused by tumor chemotherapy drugs, and the chemotherapy drugs are cisplatin.

10. The use according to claim 9, characterized in that The maslinic acid derivative is a pharmaceutically acceptable salt thereof.

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