Application of curcumin in improvement of puberty male reproductive capacity damage caused by chemotherapeutic drugs

Abstract

The invention discloses an application of curcumin in improving puberty male reproductive capacity damage caused by chemotherapeutic drugs. Aiming at the problems of testicular germ cell quantity reduction, oxidative stress injury, long-term fertility damage and the like caused by cytosine arabinoside chemotherapy, curcumin is applied while or before the cytosine arabinoside is applied. The application is realized through combined administration or a pharmaceutical composition containing curcumin and cytarabine. Experiments show that the scheme can effectively relieve testicular tissue form damage, reduce germ cell apoptosis and antagonize oxidative stress, and significantly improve the sperm quality of adult individuals. The invention provides a new potential strategy for synchronously protecting the reproductive function of a clinical child tumor patient during chemotherapy.

Description

Technical Field

[0001] This invention belongs to the field of reproductive biology technology, specifically relating to the application of curcumin in improving male reproductive capacity impairment caused by chemotherapy drugs during puberty. Background Technology

[0002] Cancer is a major public health problem that seriously threatens human health, and chemotherapy is one of its main treatment methods. Cytarabine (Ara-C), as a core chemotherapy drug for treating childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), kills rapidly proliferating cancer cells by inhibiting DNA synthesis. However, it lacks cell specificity and produces significant toxicity to normal tissues during treatment, with damage to the reproductive system being particularly prominent. This may lead to long-term risks of future fertility loss for patients, especially child survivors.

[0003] Children, especially those in pre-puberty, are in a critical developmental window for their reproductive system. During this time, testicular germ cells (such as spermatogonial stem cells) and Sertoli cells are abnormally sensitive to chemotherapy drugs. Traditionally, it was believed that the testes in children were relatively "quiet" and unaffected by damage. However, recent studies have confirmed that pre-puberty testicular tissue is highly vulnerable to drugs such as cytarabine, which can cause damage to the seminiferous tubule structure, a sharp decrease in the number of germ cells, a significant increase in oxidative stress levels, and increased apoptosis. These damages can even persistently affect spermatogenesis and fertility in adulthood. Therefore, developing an adjunctive intervention strategy that can effectively alleviate the pre-pubertal reproductive toxicity caused by chemotherapy drugs without affecting the efficacy of anti-tumor therapy has become a major problem urgently needing to be solved in the fields of clinical pediatric oncology and reproductive medicine.

[0004] Currently, clinical strategies for protecting reproductive function during chemotherapy are very limited, mainly focusing on the cryopreservation of sperm or oocytes, which is not applicable to patients in pre-puberty. Although some studies have explored the use of hormone regulators (such as GnRH agonists) or antioxidants for protection, their effectiveness is controversial and may cause additional endocrine disruption or systemic side effects. Therefore, finding a safe, efficient, and highly specific chemopreservative to synergize with chemotherapy regimens to achieve the dual goals of "anti-cancer" and "fertility preservation" has significant clinical need and social value.

[0005] Curcumin, a natural polyphenolic compound extracted from the rhizome of turmeric, has attracted considerable attention due to its remarkable antioxidant, anti-inflammatory, and anti-apoptotic activities. Studies have shown that curcumin can effectively scavenge free radicals, activate endogenous cellular antioxidant pathways (such as Nrf2 / HO-1), and stabilize mitochondrial function, thus exhibiting protective effects in various organ injury models. These properties theoretically give it the potential to become a protective agent against chemotherapeutic reproductive toxicity.

[0006] In the prior art, studies have explored the application of curcumin in reproductive system-related diseases. For example, Chinese patent application CN119632961A discloses that curcumin can be used to prevent and treat GC1 damage in mouse spermatogonia induced by triptolide, with the mechanism involving the regulation of oxidative stress and reduction of apoptosis. Another Chinese patent application CN117323291A discloses a thermosensitive hydrogel containing curcumin and dinogest for the local treatment of adenomyosis, aiming to increase the local drug concentration at the lesion site and reduce systemic side effects. In addition, Chinese patent application CN117357501A discloses the application of astaxanthin-curcumin complex in promoting ovarian function or treating polycystic ovary syndrome (PCOS), emphasizing its role in improving the reproductive endocrine environment through antioxidant and anti-inflammatory effects. However, the above-mentioned prior art focuses on reproductive damage caused by specific traditional Chinese medicine components (triptolide) or diseases of the reproductive system itself (adenomyosis, PCOS), and the damage mechanism and pathological environment are fundamentally different from the systemic, high-intensity cytotoxic damage caused by chemotherapy drugs. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides the application of curcumin in improving the damage to male reproductive capacity in adolescence caused by chemotherapy drugs. By combining curcumin with cytarabine, it is possible to effectively exert anti-tumor effects while significantly alleviating the damage to testicular reproductive function in pre-pubertal males caused by cytarabine, thus providing a potential new strategy for fertility protection for pediatric chemotherapy patients.

[0008] This invention is achieved through the following technical solution:

[0009] Application of curcumin in the preparation of drugs to improve testicular reproductive capacity impairment caused by cytarabine in pre-pubertal males.

[0010] Preferably, the damage includes an increase in the number of apoptotic cells in the seminiferous tubules, a decrease in the number of germ cells, an increase in testicular oxidative stress levels, and / or a decrease in sperm quality in adulthood.

[0011] Preferably, the drug is administered in combination with cytarabine.

[0012] Preferably, the combined administration is simultaneous administration, sequential administration, or administration as an active ingredient in the same pharmaceutical composition.

[0013] Preferably, the combined application involves applying curcumin before applying cytarabine.

[0014] A pharmaceutical composition for the combined treatment of tumors and the protection of male reproductive function in pre-pubertal men, wherein the active ingredients of the pharmaceutical composition consist of curcumin and cytarabine.

[0015] Preferably, the dosage of curcumin is 100-200 mg / kg; the dosage of cytarabine is 70 mg / kg.

[0016] Preferably, it also includes a pharmaceutically acceptable carrier or excipient.

[0017] Preferably, the curcumin and cytarabine are prepared as independent formulation units for simultaneous, separate, or sequential administration.

[0018] Preferably, the dosage form of the pharmaceutical composition is an injection, an oral preparation, or a transdermal patch.

[0019] The beneficial effects of this invention are as follows:

[0020] (1) This invention is the first to explicitly propose the use of curcumin to alleviate specific testicular reproductive damage in pre-pubescent males caused by the chemotherapy drug cytarabine. Animal experiments have confirmed that this approach can effectively alleviate tissue damage such as inhibited weight gain, reduced testicular weight, and seminiferous tubule atrophy caused by cytarabine.

[0021] (2) The combined application of curcumin in this invention can significantly antagonize the decrease in the number of germ cells in the testes caused by cytarabine. Specifically, it maintains the number of spermatogonial stem cells (PLZF positive), Sertoli cells (SOX9 positive), and other germ cells (DDX4 positive), laying the cellular foundation for subsequent normal spermatogenesis.

[0022] (3) This invention significantly reduces the level of oxidative stress caused by chemotherapy and reduces the apoptosis of germ cells by activating the Nrf2 / HO-1 endogenous antioxidant signaling pathway in testicular tissue, thereby achieving protection of testicular tissue at the molecular mechanism level.

[0023] (4) The solution of the present invention can not only alleviate immediate damage, but also have a positive impact on the reproductive function of individuals after adulthood. Experiments show that after curcumin intervention, the sperm concentration, motility and normal morphology rate of adult mice are significantly improved, indicating that its protective effect is continuous and functional.

[0024] (5) This invention creatively proposes a strategy of combining the natural active substance curcumin with the classic chemotherapy drug cytarabine. While achieving anti-tumor efficacy, this strategy aims to minimize its long-term damage to the fertility of pre-adolescent patients, providing a new and promising approach and data support for solving a major clinical challenge in pediatric cancer treatment—fertility protection. Attached Figure Description

[0025] Figure 1 The effects of curcumin on body weight and testicular morphology damage induced by cytarabine in prepubertal mice in Example 1 are shown in Figure A: Comparison of body shape among the groups of mice; Comparison of testicular size and shape among the groups of mice; Figure C: Curve of body weight changes among the groups of mice during drug administration (n=10); Figure D: Bar chart of testicular weight among the groups of mice (n=10); Figure E: Bar chart of testicular-to-body weight ratio among the groups of mice (n=10); Figure F: Representative images of hematoxylin and eosin staining of testes from each group of mice (scale bar: 50 μm); Figure G: Bar chart of seminiferous tubule diameter among the groups of mice; Figure H: Bar chart of seminiferous tubule area among the groups of mice. * P < 0.05 ** P < 0.01, *** P < 0.001, ns represents no significant difference;

[0026] Figure 2 The following is an example of the effect of curcumin on the decrease in the number of testicular germ cells induced by cytarabine in Example 2: A is a representative image of immunofluorescence staining of paraffin sections of mouse testes from each group (scale bar: 50 μm); B is a bar chart showing the number of PLZF-positive cells in the seminiferous tubules of each group of mice (n=50); C is a bar chart showing the number of SOX9-positive cells in the seminiferous tubules of each group of mice (n=50); D is a bar chart showing the number of DDX4-positive cells in the seminiferous tubules of each group of mice (n=50); E is a Western blot analysis of the expression levels of PLZF, SOX9, and DDX4 proteins in the testicular tissue of each group of mice; F is a statistical graph showing the relative expression levels of PLZF protein in the testes of each group of mice; G is a statistical graph showing the relative expression levels of SOX9 protein in the testes of each group of mice; H is a statistical graph showing the relative expression levels of DDX4 protein in the testes of each group of mice; among them, * P < 0.05 ** P < 0.01, *** P < 0.001, ns represents no significant difference;

[0027] Figure 3The effects of curcumin on cytarabine-induced oxidative stress and apoptosis in the testes of Example 3 are as follows: A shows representative images of TUNEL apoptosis staining in testicular tissue of mice in each group (red signals represent apoptotic cells, scale bar: 50 μm); B shows a statistical bar chart of the apoptotic cell rate in each group of mice (n=50); C shows the protein expression levels of representative marker molecules (Nrf2 and HO-1) in testicular tissue of mice in each group of mice detected by Western blotting; D shows a statistical graph of the relative expression level of Nrf2 protein in the testes of mice in each group of mice; E shows a statistical graph of the relative expression level of HO-1 protein in the testes of mice in each group of mice; F shows a statistical bar chart of MDA content in GC-1 cells after treatment in each group; G shows a statistical bar chart of GSH content in GC-1 cells after treatment in each group; among them, * P < 0.05 ** P < 0.01, *** P < 0.001, ns represents no significant difference;

[0028] Figure 4 The following are examples illustrating the effect of curcumin on adult sperm quality in mice treated with cytarabine in Example 4: A shows representative images of hematoxylin and eosin staining of paraffin sections of testes from each group of mice at 6 and 8 weeks; B shows representative images of computer-aided sperm analysis (CASA) and hematoxylin and eosin staining of the tail of one epididymis from each group of mice; C shows a statistical bar chart of sperm concentration from each group of mice (n=10); D shows a statistical bar chart of sperm motility from each group of mice (n=10); E shows a statistical bar chart of forward sperm motility from each group of mice (n=10); F shows a statistical bar chart of sperm abnormality rate from each group of mice (n=10); G shows representative images of sperm morphology staining from each group of mice; among them, * P < 0.05 ** P < 0.01, *** P < 0.001, ns represents no significant difference. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0030] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] The animals used in the following examples were 3-week-old male C57BL / 6 mice, purchased from the Experimental Animal Center of Nantong University. They had free access to food and water, controlled light (12 h / d), and the indoor temperature was 20~22℃.

[0033] Example 1: Protective effect of curcumin against cytarabine-induced damage to body weight and testicular tissue morphology in prepubertal mice.

[0034] 1. Animals and Grouping

[0035] Sixty 3-week-old (Postnatal day 21, PND 21) male C57BL / 6 mice were used, as follows: Figure 1 As shown in Figure A, the data is randomly divided into 6 groups (n=10), as follows:

[0036] Control group: intraperitoneal injection of normal saline;

[0037] Solvent control group (Vehicle): Intraperitoneal injection of corn oil;

[0038] The cytarabine model group (Ara-c) was administered 70 mg / kg of cytarabine via intraperitoneal injection.

[0039] Low-dose group of cytarabine + curcumin (Ara-c + CUR 100 mg / kg): 70 mg / kg cytarabine was injected 2 hours after intraperitoneal injection of 100 mg / kg curcumin.

[0040] High-dose group of cytarabine + curcumin (Ara-c + CUR 200 mg / kg): 200 mg / kg curcumin was injected intraperitoneally 2 h later, followed by 70 mg / kg cytarabine.

[0041] Curcumin control group (CUR): 200 mg / kg curcumin was injected intraperitoneally.

[0042] All groups were administered the medication continuously for 7 days (PND 22-28).

[0043] 2. Detection Indicators and Methods

[0044] Mouse body weight was recorded daily during drug administration. Mice in each group were sacrificed at PND 28 time point, and bilateral testes were harvested. Figure 1 (B) The testicular organs were weighed, and the testicular organ coefficient (testicular weight / body weight × 100%) was calculated. Subsequently, the testicular tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned in paraffin, and stained with hematoxylin and eosin (H&E). The morphology of the seminiferous tubules was observed under an optical microscope, and the diameter and area of ​​the seminiferous tubules were measured using ImageJ software (50 seminiferous tubules were counted for each sample).

[0045] 3. Experimental Results and Analysis

[0046] like Figure 1As shown, weight monitoring results indicated that, compared with the blank control group, the weight gain of mice in the Ara-c group was slower during PND 22-28; while the weight gain rate of mice in the low- and high-dose Ara-c+CUR groups was higher than that of the Ara-c group, with the high-dose group showing a more significant improvement. Figure 1 (C). Testicular weight and organ coefficient measurements revealed that, compared with the blank control group, the testicular weight of mice in the Ara-c group was significantly reduced (P < 0.001), and the organ coefficient was also significantly decreased (P < 0.001); compared with the Ara-c group, both testicular weight and organ coefficient significantly increased after treatment with curcumin. Figure 1 (D, E). H&E staining results showed that, compared with the blank control group, the Ara-c group had a significantly smaller seminiferous tubule diameter (P < 0.001), a significantly reduced number of spermatogenic cell layers (P < 0.001), and disordered tubular structure; compared with the Ara-c group, the combined curcumin treatment resulted in a larger seminiferous tubule diameter, an increased number of spermatogenic cell layers, and a more normal tissue morphology. Figure 1 (FH).

[0047] The experimental results of this embodiment show that curcumin can effectively alleviate the weight loss and testicular tissue morphology damage in prepubertal mice inhibited by cytarabine.

[0048] Example 2: Protective effect of curcumin against cytarabine-induced decrease in the number of testicular germ cells.

[0049] 1. Samples and Processing

[0050] Testicular tissue samples were taken from mice euthanized at PND 28 in Example 1 (50 seminiferous tubules were counted from each sample).

[0051] 2. Detection Method

[0052] (1) Immunofluorescence staining: Paraffin sections of testicular tissue were prepared, and after dewaxing, hydration, antigen repair, permeation, and blocking, immunofluorescence staining was performed using anti-PLZF antibody (spermatogonial stem cell marker), anti-SOX9 antibody (Supporting cell marker), and anti-DDX4 antibody (germ cell marker). The number of positive cells in 50 seminiferous tubules was observed and counted under a fluorescence microscope.

[0053] (2) Protein immunoblotting: Total protein was extracted from testicular tissue, and the protein concentration was determined by BCA method. The expression levels of PLZF, SOX9 and DDX4 proteins were detected by Western Blot, and GAPDH was used as an internal reference for relative quantitative analysis.

[0054] 3. Experimental Results and Analysis

[0055] Immunofluorescence results showed that, compared with the blank control group, the number of PLZF-positive spermatogonial stem cells, SOX9-positive Sertoli cells, and DDX4-positive germ cells in the seminiferous tubules of the testes of mice in the Ara-c group was significantly reduced (P < 0.001); while compared with the Ara-c group, the number of positive cells for each marker was significantly increased in both the low-dose and high-dose Ara-c+CUR groups, with the high-dose group showing a more significant protective effect. Figure 2 (Chinese AD).

[0056] Western blot results showed that, compared with the blank control group, the expression levels of PLZF, SOX9, and DDX4 proteins in the testicular tissue of the Ara-c group were significantly downregulated; compared with the Ara-c group, the expression levels of the above proteins were significantly upregulated after treatment with curcumin, and the effect showed a certain dose-dependent relationship. Figure 2 (EH). There were no significant differences (ns) in any indicators between the solvent control group (Vehicle) and the blank control group, and there were also no significant differences (ns) in any indicators between the CUR control group and the blank control group.

[0057] The experimental results of this embodiment show that curcumin can protect testicular germ cells and supporting cells from the cell count decrease induced by cytarabine damage.

[0058] Example 3: The effect of curcumin on improving cytarabine-induced oxidative stress and apoptosis in the testes.

[0059] 1. Samples and Processing

[0060] Same as Example 2.

[0061] 2. Detection Method

[0062] (1) TUNEL apoptosis detection: testicular tissue sections were dewaxed and hydrated, then proteinase K was added and treated at 20~37℃ for 15~30 min, TUNEL staining was performed, apoptotic cells were detected, and the apoptotic cell rate was calculated.

[0063] (2) Western blot: Detect the protein expression levels of Nrf2, a key regulator of oxidative stress, and its downstream effector molecule HO-1 in testicular tissue.

[0064] (3) Cell experiments: After treatment with mouse spermatogonial cell line GC-1 for 48 h, cells were collected, sonicated and centrifuged, and the contents of malondialdehyde (MDA) and glutathione (GSH) in the cells were detected.

[0065] 3. Experimental Results and Analysis

[0066] TUNEL staining results showed that, compared with the blank control group, the number of TUNEL-positive apoptotic cells in the testicular tissue of the Ara-c group was significantly increased (P < 0.001); while compared with the Ara-c group, the number of apoptotic cells in the low-dose and high-dose Ara-c+CUR groups was significantly reduced, with the high-dose group showing a more significant reduction effect. Figure 3 (A, B)

[0067] Western blot results showed that, compared with the blank control group, the expression levels of Nrf2 and its downstream antioxidant protein HO-1 were significantly downregulated in the Ara-c group (P < 0.001); while compared with the Ara-c group, the expression levels of both were significantly upregulated after treatment with curcumin, and in a dose-dependent manner. Figure 3 (CE).

[0068] In vitro GC-1 cell experiments showed that, compared with the blank control group, the content of lipid peroxidation product MDA in Ara-c group cells was significantly increased (P < 0.001), while the content of endogenous antioxidant GSH was significantly decreased (P < 0.001); compared with the Ara-c group, the combined treatment with curcumin significantly decreased the MDA content and significantly increased the GSH content. Figure 3 (F, G). There were no significant differences (ns) in any indicators between the solvent control group (Vehicle) and the blank control group, and there were also no significant differences (ns) in any indicators between the CUR control group and the blank control group.

[0069] The experimental results of this embodiment show that curcumin can play a protective role in the testes by activating the Nrf2 / HO-1 antioxidant pathway, reducing the accumulation of oxidative damage products, restoring the level of endogenous antioxidants, thereby inhibiting cytarabine-induced apoptosis of testicular cells.

[0070] Example 4: Long-term protective effect of curcumin on sperm quality in adult mice after cytarabine treatment.

[0071] 1. Animals and Processing

[0072] Based on the grouping and administration regimen of Example 1, mice were fed to PND 42 (6 weeks).

[0073] Based on the grouping and administration regimen of Example 1, mice were fed to PND 56 (8 weeks).

[0074] 2. Detection Method

[0075] Mice were sacrificed at PND 42 time point, and testicular tissue was collected, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The tissue was then observed and photographed under an optical microscope.

[0076] Mice were sacrificed at PND 56 time point. Testes and one epididymal tail were harvested for hematoxylin-eosin (H&E) staining and photographed. Sperm from the other epididymal tail were flushed with preheated PBS and subjected to computer-assisted sperm analysis (CASA) to determine sperm concentration, motility, and progressive motility. Sperm images were captured under a microscope. Simultaneously, sperm smears were prepared, morphologically stained, and sperm abnormality rate was calculated.

[0077] 3. Experimental Results and Analysis

[0078] After 2 and 4 weeks of recovery following drug administration, H&E staining results of paraffin sections of the testis showed ( Figure 4 In the Ara-c group (Group A), the seminiferous tubules did not show significant improvement; the tubular diameter area and the number of spermatogenic cell layers did not recover, and the tubular structure remained disordered. However, after treatment with curcumin, the seminiferous tubule diameter increased, the number of spermatogenic cell layers increased, and the tissue morphology tended to be normal.

[0079] CASA results showed that, compared with the control group, the Ara-c group had significantly lower sperm concentration, sperm motility, and progressive motility (P < 0.005), and significantly higher sperm abnormality rate (P < 0.001); while the Ara-c + CUR low-dose and high-dose groups showed significantly better results than the Ara-c group in all of the above indicators. * P < 0.05 ** P < 0.01), and the high-dose group showed a more significant improvement (P < 0.01). Figure 4 (Middle BF).

[0080] Furthermore, sperm morphology staining showed that sperm in the Ara-c group exhibited obvious abnormalities such as head deformities and tail curling, while the proportion of sperm with normal morphology significantly increased after combined treatment with curcumin. Figure 4 (F). There were no significant differences (ns) between the solvent control group (Vehicle) and the blank control group, and no significant differences (ns) were found between the CUR control group and the blank control group in any of the indicators.

[0081] The experimental results of this embodiment show that curcumin can effectively improve the damage to sperm quality caused by cytarabine in pre-pubertal mice and protect male reproductive function.

[0082] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. Application of curcumin in the preparation of drugs to improve testicular reproductive capacity damage caused by cytarabine in pre-pubertal males.

2. The application according to claim 1, characterized in that, The damage includes an increased number of apoptotic cells in the seminiferous tubules, a decreased number of germ cells, elevated levels of oxidative stress in the testes, and / or decreased sperm quality in adulthood.

3. The application according to claim 1, characterized in that, The drug is administered in combination with cytarabine.

4. The application according to claim 3, characterized in that, The combined administration refers to simultaneous administration, sequential administration, or administration as an active ingredient in the same pharmaceutical composition.

5. The application according to claim 3, characterized in that, The combined application refers to the administration of curcumin before the administration of cytarabine.

6. A pharmaceutical composition for combined treatment of tumors and protection of male reproductive function in prepubertal men, characterized in that, The active ingredients of the pharmaceutical composition consist of curcumin and cytarabine.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage of curcumin is 100-200 mg / kg; the dosage of cytarabine is 70 mg / kg.

8. The pharmaceutical composition according to claim 6, characterized in that, It also includes pharmaceutically acceptable carriers or excipients.

9. The pharmaceutical composition according to claim 6, characterized in that, The curcumin and cytarabine are prepared as independent formulation units for simultaneous, separate, or sequential administration.

10. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is an injection, an oral preparation, or a transdermal patch.

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