Use of leonurine or its salt in the preparation of a drug for preventing and treating radiation brain injury

Abstract

The application discloses a new use of Leonurine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating radiation-induced brain injury, specifically, providing a safe and effective therapeutic drug for iron death, neuroinflammation, neuron damage and lipid metabolism disorder accompanying the radiation-induced brain injury, solving the technical problem of lack of specific treatment means in the prior art, and having a wide clinical application prospect. Experiments adopt a C57BL / c mouse 30 Gy whole brain X-ray radiation model, and results show that Leonurine can significantly improve the body weight decrease and survival rate of the model mice, reduce the serum IL-6 and IFN-alpha levels, inhibit the activation of microglial cells in the cerebral cortex and hippocampus, protect and repair damaged neurons, and improve the ultrastructure of mitochondria and synapses; meanwhile, the expression of GPX4 and SLC7A11 proteins in the brain tissue is up-regulated, the GSH content is increased to inhibit iron death, and the levels of GPE, GPS and GPC related to lipid metabolism are restored.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of leonurine or its pharmaceutically acceptable salts in the preparation of medicaments for treating radiation-induced brain injury. Background Technology

[0002] Radiation-induced brain injury (RIBI) is a common complication in patients with head and neck tumors who undergo head or whole-brain radiotherapy. According to literature, the incidence of head and neck malignancies is rising annually, leading to a rapid increase in the RIBI patient population, with approximately 890,000 new cases each year, accounting for 4.6% of global malignant tumor cases. The main clinical manifestations are progressive cognitive impairment, including decreased memory, attention, and executive function; weight loss; neuroinflammatory responses; neuronal damage or loss; and ultrastructural damage to brain tissue, severely impacting patients' quality of life and ability to live independently. Currently, there are no specific antiviral drugs; supportive therapy or non-specific neuroprotective agents are mainly used, with limited efficacy and a high risk of side effects with long-term use, failing to effectively halt the progression of irreversible damage.

[0003] Leonurine (also known as SCM-198), the main active alkaloid of Herba Leonuri, has been shown to have certain neuroprotective and antioxidant effects. In a cerebral ischemia / reperfusion injury model, leonurine can alleviate neuronal damage and improve behavioral indicators by regulating SOD and MDA levels, the GABA pathway, and inhibiting the NO / NOS-mediated oxidative stress pathway. In addition, recent studies have found that leonurine can exert a protective effect in polycystic ovary syndrome (PCOS) granulosa cell models and diabetic nephropathy endothelial cell models by inhibiting ferroptosis through the SLC7A11 / GPX4 axis or the p62 / Nrf2 / HO-1 pathway (see related reports on PubMed, such as: Xiaohan Huang et al. Leonurine restrains granulosa cell ferroptosis through SLC7A11 / GPX4 axis to promote the treatment of polycystic ovary, 2025; Wu Aijun et al. Mechanism of action of leonurine in inhibiting ferroptosis of renal tubular epithelial cells by activating the p62 / Nrf2 / HO-1 signaling pathway, 2023). Meanwhile, the pathological mechanisms of radiation-induced brain injury have been confirmed to involve multiple injury processes, including oxidative stress, neuroinflammation, particularly microglia / astrocytosis activation and CD8+ T cell infiltration, ferroptosis manifested as downregulation of GPX4 and SLC7A11 expression, decreased GSH levels, and brain tissue glycerophospholipid metabolism disorders (abnormal levels of GPE, GPS, and GPC) (see Lifang Li et al., Ferrotosis in radiation-induced brain injury: roles and clinical implications, 2024; and related review literature on radiation-induced brain injury). Although the above mechanisms are well known, the prior art has not disclosed or suggested that leonurus japonicus can be used to treat radiation-induced brain injury, especially its application in radiation-induced brain injury accompanied by ferroptosis, neuroinflammation, and phospholipid metabolism disorders, particularly in neuronal loss and cognitive impairment caused by damage to the cerebral cortex and hippocampus.

[0004] Therefore, the technical problem in this field is that existing treatments cannot effectively protect against the multiple pathological mechanisms of radiation-induced brain injury, and there is a lack of a safe and effective specific drug to improve patient survival, reduce inflammatory response, inhibit ferroptosis and restore metabolic balance. Summary of the Invention

[0005] The purpose of this invention is to provide a novel use of leonurine or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention and treatment of radiation-induced brain injury.

[0006] The specific technical solution is as follows: The purpose of this invention is to provide a novel use of leonurine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating radiation-induced brain injury, particularly for radiation-induced brain injury accompanied by ferroptosis, neuroinflammation and lipid metabolism disorders, in order to address the lack of effective drugs in the prior art targeting the multiple pathological mechanisms of this disease.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides the use of leonurine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating radiation-induced brain injury.

[0008] Preferably, the radiation-induced brain injury is radiation-induced brain injury accompanied by ferroptosis.

[0009] More preferably, the ferroptosis is manifested as downregulation of GPX4 and SLC7A11 expression and decreased GSH content in brain tissue.

[0010] Preferably, the radiation-induced brain injury is radiation-induced brain injury accompanied by neuroinflammation, microglial activation and / or neuronal damage.

[0011] Preferably, the radiation-induced brain injury is radiation-induced brain injury accompanied by lipid metabolism disorder.

[0012] More preferably, the lipid metabolism disorder is manifested as abnormal levels of GPE, GPS, and / or GPC in brain tissue.

[0013] In a specific embodiment of the present invention, the drug achieves a therapeutic effect on radiation-induced brain injury by upregulating the expression of GPX4 and SLC7A11 in brain tissue and increasing GSH content, thereby inhibiting ferroptosis.

[0014] In another aspect, the present invention provides the use of leonurine or a pharmaceutically acceptable salt thereof in the preparation of a medicament having one or more of the following functions: (1) Improved weight loss and survival rate in mice with radiation-induced brain injury; (2) Reduce the levels of inflammatory factors IL-6 and IFN-α in the blood after radiation; (3) Inhibits the activation of microglia in the cerebral cortex and / or hippocampus; (4) Protect and repair the ultrastructure of neurons and tissues in the cerebral cortex and / or hippocampus; (5) Upregulates GPX4 and SLC7A11 in brain tissue; (6) Restore or regulate lipid metabolism disorders in brain tissue. Attached Figure Description

[0015] Appendix Figure 1 A mouse model of radiation-induced brain injury was established to analyze changes in body weight and mortality.

[0016] Appendix Figure 2 ELISA analysis of IL-6 and INF-α levels in mouse blood: **P<0.05, ****P<0.01.

[0017] Appendix Figure 3 Immunofluorescence analysis was performed on the expression of NEUN, CD68, IBA1, GFAP, and CD8 in the mouse cerebral cortex and hippocampus.

[0018] Appendix Figure 4 Transmission electron microscopy was used to analyze the ultrastructural features of the mouse cerebral cortex and hippocampus.

[0019] Appendix Figure 5 Western blot analysis of GPX4 and SCL7A11 expression in mouse brain tissue; GSH content was measured. **P<0.05, ****P<0.01.

[0020] Appendix Figure 6 Non-targeted metabolomics analysis of GPE, GPS, and GPC expression: **P < 0.05, ****P < 0.01 Detailed Implementation

[0021] The technical solution of the present invention is further illustrated below through specific embodiments. These embodiments are only for illustrating the present invention and are not intended to limit its scope. Equivalent substitutions or improvements made by those skilled in the art based on the technical solution of the present invention are all within the protection scope of the present invention.

[0022] Example 1: Experiment on the protective effect of leonurine on a mouse model of radiation-induced brain injury.

[0023] 1. Experimental Materials Experimental animals: Male BALB / c mice (6-8 weeks old, weight (20±2) g); Leonurine: Leonurine sulfate, purchased from Zhejiang Annovo Biopharmaceutical Co., Ltd., purity 99.77%, batch number: ANH-017-027-022-24; Radiation source: 6MV X-rays from a medical linear accelerator; Main reagents: IL-6, IFN-α ELISA kit; GPX4, SLC7A11 antibody (Abcam); GSH kit; NEUN, CD68, IBA1, GFAP, VEGF, CD8 immunofluorescence antibody, etc.

[0024] 2. Model Irradiation Method Place the mouse's head within the irradiation area (2×2cm) covering the entire brain, from the posterior eye line to the posterior ear line. 2 Inside the mouse, other parts of the body were covered with lead plates. The single irradiation dose was 30 Gy, the dose rate was 3 Gy / min, the irradiation lasted for 10 minutes, and the distance from the source to the skin was 100 cm.

[0025] 3. Experimental grouping and dosing regimen Mice were randomly divided into 8 groups (8 mice in each group). ① Blank control group (control group): BALB / c mice were administered physiological saline by gavage; ②Radiation model group (RT group): BALB / c mice were irradiated with 6MV X-rays to the whole brain, 30Gy, once, and administered with physiological saline by gavage; ③Low-dose Leonurus alkaloid group (LEO 25mg / kg): BALB / c mice were administered SCM-198 (25mg / kg) by gavage. ④ Leonurus tinctoria medium dose group (LEO 50 mg / kg): BALB / c mice were administered SCM-198 (50 mg / kg) by gavage. ⑤ High-dose Leonurus japonicus group (LEO 100 mg / kg): BALB / c mice were administered SCM-198 (100 mg / kg) by gavage. ⑥ Treatment group 1 (RT+LEO 25mg / kg): BALB / c mice were irradiated with 6MV X-rays to the whole brain, with a total dose of 30 Gy, in a single dose; SCM-198 (25mg / kg) was administered by gavage daily from 14 days before irradiation to 30 days after irradiation. ⑦ Treatment Group 2 (RT+LEO 50mg / kg): BALB / c mice were irradiated with 6MV X-rays to the whole brain, with a total dose of 30Gy, in a single dose; SCM-198 (50mg / kg) was administered by gavage daily from 14 days before irradiation to 30 days after irradiation. ⑧ Three groups of BALB / c mice (RT+LEO 100mg / kg) were treated with whole-brain irradiation with 6MV X-rays, with a total dose of 30Gy, in a single dose; SCM-198 (100mg / kg) was administered by gavage daily from 14 days before irradiation to 30 days after irradiation.

[0026] 4. Observation Indicators and Methods (1) General condition observation: Record the changes in mouse weight and mortality daily; (2) Detection of serum inflammatory factors: Blood samples were taken from the orbital cavity at 3 hours, 6 hours, 7 days and 14 days after radiation, and the serum IL-6 and IFN-α levels were measured by ELISA. (3) Immunofluorescence staining of brain tissue: samples were taken from the cerebral cortex and hippocampus, frozen sections were prepared, and double immunofluorescence staining with NEUN, CD68, IBA1, GFAP, VEGF and CD8 was performed. The fluorescence intensity was observed and quantitatively analyzed by laser confocal microscopy. (4) Transmission electron microscopy observation: Tissue from the cerebral cortex and hippocampus was routinely fixed, dehydrated, embedded, and ultrathinly sectioned. Transmission electron microscopy was used to observe the ultrastructural changes of mitochondria, synapses, etc. (5) Western blot detection: Total protein was extracted from the cerebral cortex and hippocampus, and the expression levels of GPX4 and SLC7A11 proteins were detected; (6) GSH content determination: The GSH content in the cerebral cortex and hippocampus was determined using a GSH detection kit; (7) Non-targeted metabolomics analysis: Brain tissue samples were collected and non-targeted metabolomics detection was performed using liquid chromatography-mass spectrometry (LC-MS), with a focus on analyzing the changes in the levels of glycerol phospholipid metabolism-related metabolites (GPE, GPS, GPC).

[0027] 5. Experimental Results

[0028] (1) Leonurus japonicus significantly improved the survival rate and weight loss in mice with radiation-induced brain injury. Figure 1 (See attached) Figure 1 As shown, From day 4 to 14 post-irradiation, the food and water intake, as well as body weight, of mice in the RT group decreased significantly. Treatment group 1 (RT + LEO 25 mg / kg) and treatment group 2 (RT + LEO 50 mg / kg) did not improve the decrease in food and water intake or body weight; treatment group 3 (RT + LEO 100 mg / kg) effectively improved food and water intake, and body weight recovered. From day 12 post-irradiation, the body weight of mice in the leonurine treatment group increased significantly compared to the radiotherapy group (P<0.01), and the mortality rate decreased.

[0029] (2) Leonurus japonicus significantly reduced serum inflammatory factor levels ( Figure 2The expression level of IL-6 in the serum of mice in the RT group was significantly higher than that in the control group. The IL-6 expression levels in the three treatment groups were significantly lower than those in the RT group at 6 hours and 14 days after radiation (P<0.01); there was no difference among the groups at 7 days after radiation (P>0.05). The expression level of interferon-α (INF-a) in the serum of mice in the RT group was significantly higher than that in the control group. The INF-a expression levels in the three treatment groups were significantly lower than those in the RT group at 6 hours and 14 days after radiation (P<0.01); there was no difference at 7 days after radiation (P>0.05). These findings indicate that IL-6 and INF-a are correlated with the occurrence and development of radiation-induced brain injury. Radiation increases the release of the inflammatory factor IL-6. INF-a, as a cytokine with multiple immunomodulatory and inflammatory mediating effects, can promote the recruitment and activation of inflammatory cells in radiation-induced brain injury, exacerbate the inflammatory response, expand the extent of brain tissue damage, and thus intensify brain tissue damage. Leonurine sulfate can significantly reduce the expression of inflammatory factors in radiation-induced brain injury and regulate immune and inflammatory mediators, thereby alleviating brain injury symptoms.

[0030] (3) Leonurus alkaloids inhibit RIBI-induced neuroinflammation ( Figure 3 Immunofluorescence results showed that there were differences in the fluorescence expression of CD68, IBA-1, and NEUN in the hippocampus and cortex of mice in the RT group and the treatment group 3. The expression of CD68 and IBA-1 was increased in the RT group and decreased in the treatment group 3 (P<0.05). The expression of NEUN was increased, which protected and repaired damaged neurons.

[0031] (4) Leonurus alkaloids protect the ultrastructure of brain tissue ( Figure 4Transmission electron microscopy revealed the following: ① Blood-brain barrier imaging: In the control group, endothelial cells showed no edema, the basement membrane was intact, and tight junctions were clear and intact; in the RT group, endothelial cells showed edema, and the basement membrane was ruptured; in the treatment group 2, endothelial cells showed edema, the basement membrane was partially dissolved, the mitochondrial double membrane structure of endothelial cells was destroyed, the matrix was extensively dissolved, and cristae were missing; in the treatment group 3, no edema was observed in endothelial cells, the mitochondrial double membrane structure was intact, the matrix was full, and a small number of cristae were visible. ② Neuronal and chemical synapse imaging: In the control group, neurons showed no swelling, intact structure, and intact mitochondrial structure; in the RT group, neuronal edema, structural disorder, Golgi body dissolution, partial mitochondrial swelling, partial mitochondrial atrophy, double membrane structure destruction, matrix dissolution, and reduced or absent cristae were observed; in the treatment group 2, neurons showed perinuclear edema, partial dissolution of the basement membrane, mitochondrial swelling, and disordered cristae arrangement; in the treatment group 3, fine particles were scattered within the neuronal nucleus, perinuclear Golgi bodies and rough endoplasmic reticulum were visible in the cytoplasm, mitochondria showed mild swelling, and cristae structure was intact. ③ Astrocyte imaging: In the control group, cells showed mild swelling and intact mitochondrial structure; in the RT group, vacuolation, matrix edema, large-area cytoplasmic dissolution, loss of organelles, nuclear membrane dissolution, and massive chromatin condensation were observed; in the treatment group 2, cells showed sparse cytoplasm, occasional destruction of the mitochondrial double membrane structure, and matrix dissolution; in the treatment group 3, cytoplasm was slightly sparse, mitochondria were well repaired, and the double membrane structure was intact.

[0032] (5) Leonurus alkaloids upregulate GPX4 / SLC7A11 expression and increase GSH content, inhibiting ferroptosis. Figure 5 Western blot results showed that 6 hours after whole-brain irradiation in mice, there was no difference in the expression of GPX4 and SCL7A11 between the RT group and the treatment group (P>0.05); 7 days after irradiation, the expression of GPX4 and SCL7A11 in both groups was significantly lower than that in the control group, but there was still no difference in expression between the two groups (P>0.05); 14 days after irradiation, the expression of GPX4 and SCL7A11 in the RT group further decreased, while the expression of GPX4 and SCL7A11 in the treatment group was significantly higher than that in the radiotherapy group (P<0.01), and gradually returned to the level of the control group (P>0.05).

[0033] (6) Leonurus alkaloids regulate lipid metabolism disorders ( Figure 6Untargeted metabolomics analysis showed that the levels of GPE, GPS, and GPC in brain tissue were significantly reduced in the RT group; the levels of these metabolites were significantly increased in the treatment group (**P<0.01, *P<0.05), indicating that leonurine can improve radiation-induced lipid metabolism disorders. Thirty days after radiation, untargeted metabolomics data showed differential expression of phosphatidylethanolamine (GPE), glycerophosphoserine (GPS), and glycerophosphocholine (GPC) in both the RT and treatment groups, with GPC showing the highest differential expression.

[0034] Example 2: Dose-response relationship of different doses of leonurine Using the same radiation model as in Example 1, three groups of leonurine were established: a low-dose group (25 mg / kg), a medium-dose group (50 mg / kg), and a high-dose group (100 mg / kg). Administration began 14 days before radiation and continued until 30 days after radiation. Results showed that the 100 mg / kg group was significantly better than the model group in terms of survival rate, weight improvement, reduction of inflammatory factors, and upregulation of GPX4 / SLC7A11 expression. Furthermore, the 100 mg / kg group approached a saturation effect, suggesting that 100 mg / kg is the optimal dose.

[0035] Example 3: Preparation of Leonurus alkaloid sustained-release capsules Take 200g of leonurine raw material, mix it evenly with 300g of hydroxypropyl methylcellulose (sustained-release material) and 100g of lactose, add an appropriate amount of binder to granulate, dry and then fill into capsules, each containing 200mg of leonurine. This sustained-release capsule is suitable for long-term prevention of chronic radiation-induced brain injury, with a release time of more than 12 hours, and a dosage of 100mg / kg per day. The drug should be administered within 24 hours after the radiation event.

Claims

1. Use of a leonurine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating radiation-induced brain injury.

2. The use according to claim 1, wherein the radiation-induced brain injury is radiation-induced brain injury accompanied by neuroinflammation, microglial activation and / or neuronal damage.

3. The use according to claim 1, wherein the radiation-induced brain injury is radiation-induced brain injury accompanied by ferroptosis.

4. The use according to claim 3, wherein the radiation-induced brain injury is characterized by downregulation of GPX4 and SLC7A11 expression and decreased GSH content in brain tissue.

5. The use according to claim 1, wherein the radiation-induced brain injury is radiation-induced brain injury accompanied by lipid metabolism disorder.

6. The use according to claim 5, wherein the lipid metabolism disorder is manifested as abnormal levels of GPE, GPS, and / or GPC metabolism in brain tissue.

7. The use according to claim 1, wherein the radiation-induced brain injury is radiation-induced brain injury accompanied by mitochondrial damage.

8. The use according to any one of claims 1-8, wherein the drug is an oral sustained-release formulation, and the radiation-induced brain injury is acute, early delayed, or late delayed brain injury caused by radiotherapy for head and neck tumors.

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