Application of isoliquiritin in preparation of medicine for preventing and treating central nervous injury caused by mustard gas

Abstract

The invention relates to the technical field of medicines, in particular to application of isoliquiritin in preparation of medicines for preventing and treating mustard gas nerve injury. The invention also provides an application of isoliquiritin in preparation of neuroprotective drugs. The isoliquiritin exerts effective neuroprotective activity on mustard gas injury, can remarkably improve the situation that the survival rate of PC12 cells is reduced due to mustard gas, remarkably inhibits LDH outflow and ROS rising of the cells, and relieves cell DNA injury caused by mustard gas. At the animal level, mouse brain tissue neuronal degeneration and nissl body reduction caused by mustard gas are improved, and the spontaneous activity condition of mouse brain tissue neuronal degeneration and nissl body reduction are improved. The invention provides a new reference way for treating mustard gas nerve injury, and the isoliquiritin has a prospect of preparing a neuroprotective agent aiming at mustard gas.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of isoglycyrrhizin in the preparation of drugs for preventing and treating central nervous system damage caused by mustard gas. Background Technology

[0002] Sulfur mustard (SM), also known as 2,2'-dichlorodiethyl sulfide, is a typical vesicant chemical agent. Through alkylation, it denatures the structures of biological macromolecules such as DNA and proteins, causing a series of tissue damages. Due to its potent toxicity, large lethality, ease of production and synthesis, and difficulty in prevention and treatment, it is known as the "king of poisons" and has been used extensively in past conflicts. As a significant component of abandoned chemical weapons, mustard gas causes long-term damage to the skin and nervous system of victims. Furthermore, its quantity and location are unknown, and harm can occur at any time, posing a serious threat.

[0003] The brain is one of the target organs for mustard gas damage. Studies have found that skin exposure to mustard gas affects the brain rapidly, taking only 10 minutes in rats, and the brain's high lipid content facilitates mustard gas retention. A growing number of clinical studies report the neurotoxic effects of mustard gas, primarily manifesting as anxiety, depression, headache, insomnia, cognitive impairment, convulsions, epilepsy, and muscle atrophy. Research suggests that mustard gas may accelerate neurodegeneration or cause permanent neurological dysfunction through DNA hydrocarbonation damage, inducing oxidative stress and apoptosis, and leading to neuronal demyelination.

[0004] Currently, there are no specific antidotes for mustard gas poisoning and its induced nerve damage. Clinical treatment mainly involves symptomatic relief with existing chemical drugs, but the results are not ideal. Licorice (Glycyrrhizae Radix et Rhizoma) is one of the most commonly used traditional Chinese medicines in clinical practice, and it has long been known for its excellent properties of antagonizing drug toxicity and neuroprotection. Isoglycyrrhizin is an important flavonoid active component in licorice, possessing not only anti-inflammatory, antifungal, and antioxidant activities, but also excellent anti-neurotoxic and neuroprotective effects. It has been reported that isoliquiritigenin and drug combinations containing isoliquiritigenin can be used to treat systemic lupus erythematosus (Chinese patent document CN118910231A), osteoporosis (Chinese patent document CN114848659A), ischemic stroke (Chinese patent document CN111253453A), depression (Chinese patent document CN120131683A), silicosis (Chinese patent document CN119818516A), asthma (Chinese patent document CN115089598A), and colon cancer (Chinese patent document CN112546086A), etc. However, there are no related patents or literature reports on the use of isoliquiritigenin to alleviate central nervous system damage caused by mustard gas toxicity. Summary of the Invention

[0005] The purpose of this invention is to provide the application of isoglycyrrhizin in the preparation of neuroprotective drugs. This invention also provides the application of isoglycyrrhizin in the preparation of drugs for preventing and treating mustard gas-induced nerve damage.

[0006] In this invention, isoglycyrrhizin exhibits significant neuroprotective activity against nerve cells exposed to mustard gas, significantly inhibiting the decline in PC12 cell survival induced by mustard gas, and showing significant inhibitory effects on mustard gas-induced DNA damage and ROS accumulation. In this invention, isoglycyrrhizin also demonstrates significant neuroprotective activity against mice exposed to mustard gas through the skin, improving the reduction in spontaneous activity caused by mustard gas poisoning and alleviating pathological changes in mouse brain tissue.

[0007] Based on the above technical solution, in a first aspect of the present invention, isoglycyrrhizin is provided for the application of isoglycyrrhizin in the preparation of drugs for preventing and treating nerve damage caused by mustard gas, wherein the chemical structure of isoglycyrrhizin is shown in Formula I below:

[0008]

[0009] Formula I.

[0010] Furthermore, the aforementioned drug for preventing and treating nerve damage caused by mustard gas is a drug for preventing and treating central nervous system damage caused by mustard gas.

[0011] Furthermore, the drugs for preventing and treating nerve damage caused by mustard gas include drugs for preventing and treating headaches, neurodegenerative diseases, anxiety, depression, convulsions, or epilepsy caused by mustard gas poisoning.

[0012] Furthermore, isoliquiritigenin can reduce mustard gas-induced DNA damage, apoptosis, and structural damage in nerve cells, reduce oxidative stress damage caused by mustard gas exposure, reduce anxiety and depression-like behaviors, reduce mustard gas damage to the central nervous system, and help maintain the normal morphology and function of neurons.

[0013] A second aspect of the invention provides the use of isoglycyrrhizin in the preparation of neuroprotective drugs.

[0014] Furthermore, the neuroprotective drug is a drug used for neurological diseases such as headache, anxiety, depression, or neurodegenerative diseases.

[0015] In a third aspect, the present invention provides a medicament for preventing and treating nerve damage caused by mustard gas, wherein the medicament is composed of isoglycyrrhizin as the sole active ingredient, or isoglycyrrhizin is used in combination with other drugs to form a pharmaceutical composition.

[0016] Furthermore, the drug also includes one or more pharmaceutical excipients to improve drug absorption or facilitate administration, such as in pharmaceutically suitable dosage forms like powders, tablets, capsules or pills, oral liquids, or injections.

[0017] The advantages of this invention are:

[0018] Currently, there is no specific antidote for mustard gas, a chemical weapon, and it poses a significant real threat as a key component of abandoned chemical weapons. The brain is a major target organ for mustard gas, and poisoning can lead to a range of neuropsychiatric symptoms, including anxiety, depression, and cognitive impairment. In this invention, isoglycyrrhizin exhibits effective neuroprotective activity against mustard gas damage, significantly improving the reduced survival rate of PC12 cells caused by mustard gas, significantly inhibiting cellular LDH efflux and ROS elevation, and mitigating mustard gas-induced cellular DNA damage. In animal studies, it improves mustard gas-induced neuronal degeneration and Nissl body reduction in mouse brain tissue, and enhances spontaneous activity. This invention provides a novel approach for the treatment of mustard gas-induced neurological injury, and isoglycyrrhizin shows promise in the development of neuroprotective agents against mustard gas. Attached Figure Description

[0019] Figure 1 A shows the effect of isoglycyrrhizin on the viability of PC12 cells after mustard gas exposure. B shows the effect of isoglycyrrhizin on the efflux of lactate dehydrogenase (LDH) from PC12 cells after mustard gas exposure.

[0020] Figure 2 Figure A shows the effect of isoglycyrrhizin on DNA damage levels in cells exposed to mustard gas. γ-H2AX is a marker of cellular DNA damage. Figure B shows the effect of isoglycyrrhizin on oxygen free radical (ROS) levels in cells exposed to mustard gas.

[0021] Figure 3 A shows the effect of isoglycyrrhizin on the open field behavior of mice exposed to mustard gas. B shows the effect of isoglycyrrhizin on the neuronal pathological changes in the brain tissue of mice exposed to mustard gas. Detailed Implementation

[0022] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0023] Example 1: Effect of isoglycyrrhizin on the survival rate of PC12 nerve cells after mustard gas exposure

[0024] PC12 cells in logarithmic growth phase were fed at a rate of 2 × 10⁻⁶. 5Cells were seeded at a density of 100 μL / well in 96-well plates. After incubation at 37°C for 24 h, the mustard gas stock solution was diluted to a final concentration of 10 μM with serum-free DMEM medium and PBS, replacing the DMEM complete medium in the model group and drug-treated groups. After incubation at 37°C for 30 min, the drug-treated solution was discarded. The model group was replaced with fresh medium, while the drug-treated groups were added with DMEM complete medium containing final concentrations of 0.1, 1, 10, and 100 μM isoglycyrrhizin, respectively, and cultured for another 24 h. The supernatant was removed from each group, and DMEM complete medium containing 10 μL of CCK8 solution was added to each well, and incubation was carried out for 1 h. The absorbance of each well was measured at 450 nm using a microplate reader to calculate cell viability.

[0025] The results are as follows Figure 1 As shown in Figure A, compared with normal cells in the control group, mustard gas exposure significantly reduced the survival rate of PC12 cells. 1-100 μM isoglycyrrhizin dose-dependently increased the survival rate of PC12 cells, with 100 μM isoglycyrrhizin increasing the cell survival rate from (47.25±4.39)% to (84.79±6.28)%, demonstrating a significant protective effect on neurons.

[0026] Example 2: Effect of isoglycyrrhizin on LDH efflux from PC12 cells after mustard gas exposure

[0027] PC12 cells in logarithmic growth phase were fed at a rate of 2 × 10⁻⁶. 5 Mustard gas was seeded at a density of 100 μL / well in 96-well plates, with cell-free wells and control wells containing maximum enzyme activity that were not treated with the drug or exposed to the drug. After incubation at 37°C for 24 h, the mustard gas stock solution was diluted to a final concentration of 10 μM with serum-free DMEM medium and PBS, replacing the DMEM complete medium in the model group and drug-treated groups. After incubation at 37°C for 30 min, the drug-treated solution was discarded. The model group was replaced with fresh medium, while the drug-treated groups were added with DMEM complete medium containing final concentrations of 0.1, 1, 10, and 100 μM isoglycyrrhizin, respectively, and cultured for another 24 h. 10 μL of LDH release agent Lysis Buffer was added to the blank control group, mixed by pipetting, and cultured in a cell culture incubator for 30 min. During the preparation of LDH working solution: 2 mL each of lactic acid solution, 1×INT solution, and enzyme solution, totaling 6 mL, were mixed well. 60 μL of LDH working solution was added to each well. The 96-well plate was wrapped with aluminum foil and incubated at room temperature with gentle shaking on a shaker for 30 min. The absorbance of each well was measured at 490 nm using a microplate reader. Cell damage rate = (sample well absorbance - control absorbance) / (absorbance of maximum enzyme activity - control absorbance) × 100%.

[0028] The results are as follows Figure 1As shown in Figure B, compared with normal cells in the control group, mustard gas exposure caused severe damage to PC12 cells, with a significant increase in LDH efflux. After administration of isoliquiritigenin, apoptosis and structural damage were significantly improved in the treatment group, and the amount of LDH efflux decreased with increasing isoliquiritigenin concentration. Isoliquiritigenin at levels of 1-100 μM significantly reduced LDH leakage during neuronal damage, indicating that it plays a protective role in the structural integrity of PC12 cells.

[0029] Example 3: Detection of the effect of isoliquiritigenin on DNA damage in cells exposed to mustard gas using γ-H2AX immunofluorescence assay

[0030] Aspirate the culture medium and wash once with PBS. Add 1 mL of fixative to each well, aspirate the fixative after 10 min, and wash three times with washing buffer for 3-5 min each time, aspirating all washing buffer after the last wash. Add 1 mL of immunostaining blocking buffer, incubate at room temperature for 20 min, aspirate the blocking buffer, add 1 mL of γ-H2AX rabbit monoclonal antibody, and incubate at room temperature for 1 h. Wash three times with washing buffer for 5-10 min each time, aspirating all washing buffer after the last wash. Add 1 mL of anti-rabbit 488 and incubate at room temperature for 1 h. Wash twice with washing buffer for 5-10 min each time. Add 1 mL of nuclear staining solution (DAPI) and stain at room temperature for 5 min. Aspirate the DAPI staining solution and wash three times with washing buffer for 3-5 min each time. Add an appropriate amount of anti-fluorescence quenching mounting solution, mount with a coverslip, and observe under a fluorescence microscope. γ-H2AX staining shows green fluorescence, and DAPI staining of the cell nucleus shows blue fluorescence.

[0031] The results are as follows Figure 2 As shown in Figure A, compared with normal cells, the number of DAPI-positive viable cells was significantly reduced and the number of γ-H2AX-positive DNA-damaged cells was significantly increased in the mustard gas exposure group. Compared with the poisoning group, the number of viable cells was significantly increased and the number of γ-H2AX-positive DNA-damaged cells was significantly reduced in the isoglycyrrhizin treatment group, indicating that isoglycyrrhizin can alleviate mustard gas-induced DNA damage.

[0032] Example 4: Detection of the effect of isoliquiritigenin on ROS levels in mustard gas-exposed cells using the DCFH-DA probe method

[0033] PC12 cells were seeded in 6-well plates one day in advance. Mustard gas stock solution was diluted to 10 μM with serum-free medium and PBS, replacing the DMEM complete medium in the model and drug-treated groups. After incubation at 37°C for 30 min, the disinfectant was discarded. The model group was replaced with fresh medium, while the drug-treated group was added to DMEM complete medium containing 10 μM isoglycyrrhizin, and cultured for another 24 h. The medium in each well was then removed, and 10 μM DCFH-DA was dissolved in serum-free medium according to the manufacturer's instructions. 2 mL of the DCFH-DA dilution was added to each well. Cells were incubated at 37°C in the dark for 20 min, washed with PBS to remove residual liquid, and observed and imaged under a fluorescence microscope.

[0034] The results are as follows Figure 2 As shown in Figure B, compared with normal cells, the fluorescence intensity of ROS in the mustard gas-exposed cells was significantly increased; after treatment with 10 μM isoglycyrrhizin, the ROS content was significantly reduced. Isoglycyrrhizin can alleviate oxidative stress damage induced by mustard gas exposure.

[0035] Example 5: Open field test to evaluate the effect of isoglycyrrhizin on spontaneous activity in mice after mustard gas exposure.

[0036] Healthy male C57BL / 6 mice were randomly divided into three groups: a blank control group (injected with an equal volume of solvent), a mustard gas exposure group, and an isoglycyrrhizin treatment group. The isoglycyrrhizin treatment group received an intravenous injection of 10 mg / kg of isoglycyrrhizin solution prepared with physiological saline within 30 minutes after mustard gas exposure, at a volume of 0.1 ml / 10 g. The mice underwent an open field experiment 24 hours later. Two hours before the experiment, the mice were moved to an area near the open field to allow them to acclimatize. At the start of the experiment, the mice were placed in the box from the upper left corner. Each mouse was allowed to move freely in the open field for 5 minutes, and their behavior and movement trajectory were monitored and recorded in real time. After each experiment, the mice were cleaned with 75% ethanol to remove any odor or information left by the previous mouse. The time spent in the central area, the total distance traveled, and the speed of movement were recorded.

[0037] The results are as follows Figure 3 As shown in the representative trajectory diagram in Figure A, the control group mice exhibited active spontaneous activity, with a large number of movement trajectories covering the central and surrounding areas of the open field. In contrast, the mice in the poisoned group showed significantly reduced spontaneous activity, shorter movement distances, and mostly stayed on the periphery of the field, rarely venturing into the central area for exploration. Mice treated with 10 mg / kg isoglycyrrhizin showed increased exploration in the central area and improved activity levels. This suggests that central nervous system damage caused by mustard gas exposure may induce anxiety and depression-like behaviors in mice, and isoglycyrrhizin treatment can alleviate these symptoms.

[0038] Example 6: Effects of HE and Nissl staining on pathological changes in brain tissue of mice exposed to mustard gas.

[0039] Healthy male C57BL / 6 mice were randomly divided into three groups: a blank control group (injected with an equal volume of solvent), a mustard gas exposure group, and an isoglycyrrhizin treatment group. The isoglycyrrhizin treatment group received an intravenous injection of 10 mg / kg of isoglycyrrhizin solution prepared with physiological saline within 30 minutes of mustard gas exposure, at a volume of 0.1 ml / 10 g. After 24 hours, the mice were fixed, and their hearts were exposed by thoracotomy. Physiological saline was slowly injected along the heart axis. Once the heart slightly enlarged, the right atrial appendage was punctured to begin perfusion. The mice were first flushed with approximately 40 ml of physiological saline, followed by perfusion and fixation with approximately 20 ml of 4% paraformaldehyde. Successful perfusion was indicated by limb tremors, an erect tail, and a whitening of the liver. The mice were decapitated, and their brain tissue was collected and fixed.

[0040] Paraffin sections of mouse brain tissue were prepared. The brain tissue was fixed in a fixative solution for 24 h and then placed in a dehydration chamber. The brain tissue was dehydrated in ethanol solutions of varying concentrations, from low to high. The tissue blocks were then immersed in a 1:1 mixture of paraffin and xylene and incubated at 55 °C for 15 min; this was followed by incubation twice with pure paraffin, 15 min each time. A certain volume of paraffin solution was injected into an embedding frame, and the tissue was carefully placed into the frame and allowed to cool and fix. The paraffin-embedded tissue blocks were cooled on a cryostat, sectioned at a thickness of 4 μm using a microtome, and the sections were flattened using a spreader. The sections were then baked at 60 °C for 2 h. After preparation, the sections were stored at room temperature for later use.

[0041] Paraffin sections were sequentially immersed in xylene twice (15 min each time), then in anhydrous ethanol twice (5 min each time), and finally in 75% ethanol for 5 min, followed by dewaxing and hydration, and washing with PBS. They were then stained with hematoxylin for 5 min, followed by rinsing with sufficient distilled water. Differentiation was performed with 1% hydrochloric acid-alcohol solution for 5 s, followed by washing with distilled water for 5 min. Hematoxylin blueing solution was applied for 50 s, followed by washing with distilled water for 3 min. Finally, they were stained with eosin for 8 min. The stained sections were then sequentially immersed in anhydrous ethanol three times (5 min each time), and then in xylene twice (5 min each time). Finally, they were mounted with neutral resin and observed under a microscope.

[0042] Paraffin sections were sequentially immersed in xylene twice (15 min each time), then in anhydrous ethanol twice (5 min each time), and finally in 75% ethanol for 5 min, followed by dewaxing and hydration, and washing with PBS. They were then stained in Nissl stain for 5 min, followed by rinsing with sufficient distilled water. Differentiation was achieved with 1% hydrochloric acid-ethanol solution for 5 s, followed by washing with distilled water for 5 min. The stained sections were then sequentially immersed in anhydrous ethanol three times (5 min each time), and then in xylene twice (5 min each time). Finally, they were mounted with neutral resin and observed under a microscope.

[0043] The results are as follows Figure 3 As shown in Figure B. HE staining results: In the control group mice, hippocampal DG region neurons were densely packed, with tightly packed and clearly visible nuclei. In the mustard gas-treated group mice, the number of degenerated neurons in the DG region of the brain tissue was significantly increased, with many neuronal nuclei showing swelling, cell condensation, and deep staining. Compared with the model group, the number of degenerated neurons in the isoglycyrrhizin-treated group mice was significantly reduced. Nissl staining results: In the control group mice, hippocampal CA3 region neurons had numerous and clearly morphologically distinct Nissl bodies; in the mustard gas-treated group mice, CA3 region neurons showed degenerated neurons and a decrease in Nissl bodies; in the 10.0 mg / kg isoglycyrrhizin-treated group mice, the cortical tissue structure was relatively dense and clear, with slightly fewer Nissl bodies than the control group, but compared with the poisoned group, the number and morphology were significantly improved, indicating that isoglycyrrhizin treatment can alleviate the damage of mustard gas to the central nervous system of mice and help maintain the normal morphology and function of neurons.

[0044] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. The application of isoliquiritigenin in the preparation of drugs for preventing and treating nerve damage caused by mustard gas, characterized in that, The chemical structure of the isoglycyrrhizin is shown in Formula I: Formula I.

2. The application of isoliquiritigenin according to claim 1 in the preparation of drugs for preventing and treating nerve damage caused by mustard gas, characterized in that, The aforementioned drug for preventing and treating nerve damage caused by mustard gas is a drug for preventing and treating central nervous system damage caused by mustard gas.

3. The application of isoliquiritigenin according to claim 1 in the preparation of drugs for preventing and treating nerve damage caused by mustard gas, characterized in that, The drugs mentioned for preventing and treating nerve damage caused by mustard gas include drugs for preventing and treating headaches, neurodegenerative diseases, anxiety, depression, convulsions, or epilepsy caused by mustard gas poisoning.

4. The application of isoliquiritigenin according to claim 1 in the preparation of drugs for preventing and treating nerve damage caused by mustard gas, characterized in that, Isoliquiritigenin reduces mustard gas-induced DNA damage, apoptosis, and structural damage in nerve cells, reduces oxidative stress damage caused by mustard gas exposure, reduces anxiety and depression-like behaviors, reduces mustard gas damage to the central nervous system, and helps maintain the normal morphology and function of neurons.

5. Application of isoglycyrrhizin in the preparation of neuroprotective drugs.

6. The application of isoliquiritigenin according to claim 5 in the preparation of neuroprotective drugs, characterized in that, The neuroprotective drugs mentioned are used for headaches, anxiety, depression, or neurodegenerative diseases.

7. A drug for preventing and treating nerve damage caused by mustard gas, characterized in that, The drug is a pharmaceutical composition consisting of isoglycyrrhizin as the sole active ingredient or in combination with other drugs.

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