Memantine, hydrogen-rich water and application of combination of memantine and hydrogen-rich water in preparation of medicine for treating radiation-induced brain injury

Abstract

The invention relates to application of memantine, hydrogen-rich water and combination of memantine and hydrogen-rich water in preparation of a medicine for treating radiation brain injury, and the concentration of hydrogen-rich water is larger than or equal to 0.5 mmol / L. According to the invention, hydrogen-rich water and memantine are combined, so that the free radical scavenging capability level of antioxidant glutathione (GSH) and superoxide dismutase (SOD) in brain tissues can be improved, and malondialdehyde (MDA) which is a key by-product of an oxidation reaction can be inhibited; the memantine, the hydrogen-rich water and the combination of the memantine and the hydrogen-rich water in brain tissues of rats with radiation brain injury can inhibit expression of inflammatory factors and improve oxidative stress; the expression of NLRC4, Caspase-1 and Aim2 genes in the hippocampus of a rat with radiation brain injury can be inhibited; the NLRP3, NLRC4 and Caspase-1 pathways can be blocked, and the brain injury induced by the ionizing radiation can be improved.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of memantine, hydrogen-rich water and their combination in the preparation of drugs for treating radiation brain injury. Background Art

[0002] Radiotherapy is the main treatment for head and neck tumors. At the same time, patients with brain metastases often receive whole brain irradiation to treat extensive metastases, progression after radiotherapy, or metastasis to the leptomeninges. The radiotherapy area always inevitably includes surrounding normal tissues. Therefore, more than 50% of brain tumor patients who survive for more than 6 months after whole brain radiotherapy will have irreversible cognitive impairment; brain radiation brain injury (RIBI) has also become the main dose-limiting adverse event of radiotherapy, which seriously affects the quality of life. In addition, brain radiation brain injury also occasionally occurs in ionizing radiation accidents; ionizing radiation can produce toxic hydroxyl free radicals, stimulating the production of a large amount of endogenous reactive oxygen species; studies have shown that increased ROS can induce cell pyroptosis. In the classic cell pyroptosis pathway, inflammasomes such as NLRP3, NLRC4, and AIM2 are activated, and Pro-Caspase-1 is activated and cleaved to form active Caspase-1. In addition, Sung et al. found that ionizing radiation can cause upregulation of pyroptosis-related genes such as NLRP3, Caspase-1, IL-1β, and GSDMD in the lungs of mice, as well as upregulation of pulmonary fibrosis-related genes. Liu and Gao et al. found that γ-ray irradiation can activate inflammasomes and cause cell pyroptosis. Liao et al. confirmed through in vivo and in vitro experiments that ionizing radiation can cause an increase in ROS in glial cells, promote the cleavage of Caspase-1 and the release of proinflammatory cytokines IL-18 and IL-1β.

[0003] However, since the mechanism of radiation brain injury is relatively complex and the relevant mechanism is not fully understood, the clinical drug selection is limited. At present, some guidelines recommend the use of steroids and bevacizumab for treatment, but these drugs have the risk of adverse reactions. To solve these problems, the newly developed drug hydrogen-rich water has been proven to have the effect of preventing radiation brain injury, but its effect still has great room for improvement. Therefore, the development of new drugs for the prevention and treatment of radiation brain injury or pharmaceutical compositions combined with hydrogen-rich water is crucial to improve the therapeutic effect of radiation brain injury. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects in the prior art and provide the use of memantine, hydrogen-rich water and their combination in the preparation of a drug for treating radiation-induced brain injury, which can achieve the purpose of treating radiation-induced brain injury by blocking the NLRP3 / NLRC4 / Caspase-1 cell pyroptosis pathway.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A pharmaceutical composition for treating radiation brain injury, comprising an active ingredient a; the active ingredient a is memantine and its salt;

[0007] As a further technical solution, the salt includes hydrochloride.

[0008] As a further technical solution, the memantine and its salt include memantine (1-amino-3,5-dimethyladamantane amine) or memantine hydrochloride (1-amino-3,5-dimethyladamantane amine hydrochloride).

[0009] As a further technical solution, an active ingredient b is also included; the active ingredient b is hydrogen-rich water.

[0010] As a further technical solution, the concentration of the hydrogen-rich water is greater than or equal to 0.5 mmol / L, and the human dosage converted based on body surface area is approximately 0.1-0.5 mg / kg.

[0011] As a further technical solution, the pharmaceutical composition for treating radiation brain injury further includes pharmaceutically acceptable adjuvants and / or excipients.

[0012] As a further technical solution, a pharmaceutical preparation comprising the pharmaceutical composition includes tablets, capsules, powders, syrups, solutions, suspensions or aerosols.

[0013] Application of memantine and its salt or memantine and its salt in combination with hydrogen-rich water in the preparation of medicine for treating radiation brain injury.

[0014] As a further technical solution, memantine and its salts or memantine and its salts combined with hydrogen-rich water can block cell pyroptosis in the NLRP3, NLRC4, and Caspase-1 pathways.

[0015] As a further technical solution, 1) hydrogen-rich water, memantine and their combination can improve the ability of antioxidants glutathione (GSH) and superoxide dismutase (SOD) in brain tissue to scavenge free radicals, and can inhibit the key byproduct of oxidation reaction, malondialdehyde (MDA), that is, memantine, hydrogen-rich water and their combination can inhibit the expression of inflammatory factors and improve oxidative stress in the brain tissue of rats with radiation brain damage; 2) hydrogen-rich water, memantine and their combination can inhibit the expression of NLRC4, Caspase-1 and Aim2 genes in the hippocampus of rats with radiation brain damage; 3) hydrogen-rich water, memantine and their combination can block the NLRP3, NLRC4 and Caspase-1 pathways, and improve brain damage induced by ionizing radiation.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The hydrogen-rich water and memantine of the present invention interact with each other, and their combined use can exert a synergistic effect, can effectively alleviate radiation brain damage after whole-brain irradiation, can inhibit pyroptosis of brain hippocampal tissue cells, and improve the therapeutic effect of radiation brain damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The changes of Celsius volume in rats after ionizing irradiation;

[0019] Figure 2 The pictures show the pathological histological changes of CA area in rats of different groups after ionizing radiation;

[0020] Figure 3 Effects of hydrogen-rich water, memantine and their combination on inflammatory factors and oxidative stress indicators;

[0021] exist Figure 3 In, a: MDA; b: SOD; c: radicals; d: GSH;

[0022] Figure 4 Figure 2 is the mRNA expression level of pyroptosis pathway genes in rats of different groups after ionizing radiation (n=5);

[0023] exist Figure 4 Middle, a: NLRP3; b: NLRC4; c: Caspase 1; d: relative expression level of Aim2; e: IL-18; f: IL-1β;

[0024] Figure 5 This is the immunohistochemistry of NLRP3 protein;

[0025] Figure 6 This is the analysis diagram of NLRP3 protein immunohistochemistry;

[0026] exist Figure 6 Middle, a: CA1; b: CA3; c: DG;

[0027] Figure 7 This is the analysis diagram of IL-1β protein immunohistochemistry;

[0028] Figure 8 It is the protein band diagram of related proteins NLRP3, NLRC4, Caspase1, ASC, and IL-18 protein;

[0029] Fig. 9 This is a graph showing the effects of hydrogen-rich water, memantine and their combination on the expression levels of NLRP3, NLRC4, ASC, Caspase1, Cleaved Caspase1 and IL-18 proteins;

[0030] exist Fig. 9Middle, a: NLRP3; b: NLRC4; c: ASC; d: Caspase1; e: Cleaved Caspase1; f: IL-18. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0033] 1) The hydrogen-rich water cup was purchased from Shanxi Zhongfu Nuclear Instrument Co., Ltd. The product model is ZFFQ-500, and the concentration of hydrogen-rich water is greater than 0.5mmol / L;

[0034] 2) SD rats were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.;

[0035] Unless otherwise specified, the raw materials used in the present invention are commercially available.

[0036] Embodiment 1:

[0037] 1. Handling of Experimental Animals

[0038] 43 male SPF Sprague-Dawley rats (3 rats were eliminated when grouping), 5-7 weeks old, weighing 210-240 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., China. After 7 days of adaptive feeding, the rats were divided into groups. After grouping, they were irradiated for 3 days. The animal weight was 290-330 g during irradiation. The temperature, humidity, and light intensity of the animal laboratory were controllable, with a day-night ratio of 12:12 (day: 8:00-18:00; night: 18:00-8:00 the next day), and the animals were free to eat. All operations followed the relevant experimental animal ethics requirements.

[0039] Grouping:

[0040] Animals were randomly divided into 5 groups according to block groups, with 8 rats in each group, namely: control group (Control), irradiation group (IR), memantine hydrochloride group (IR+Memantine), hydrogen-rich water group (IR+HRW), and joint group (IR+Joint). All animals in each group were gavaged once a day from the third day before irradiation to the fifth day after irradiation (30 minutes before irradiation on the irradiation day). The control group and the model group were gavaged with pure water, the memantine group was gavaged with 2.5 mg / kg memantine hydrochloride (converted according to Meeh's formula body surface area), the hydrogen-rich water group was gavaged with hydrogen-rich water (hydrogen concentration not less than 1000 ppb), and the joint group used hydrogen-rich water to prepare memantine (2.5 mg / kg). The gavage volume of each group was 20 mL / kg. All rats in each group were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital, and then irradiated with X-rays at a dose rate of 300 MU / min in a prone position with their backs facing the machine head. Except for the control group, all groups received a total dose of 25 Gy whole head irradiation. The food intake of rats before and after irradiation was recorded, and the changes in food intake of rats after irradiation were statistically analyzed. The results are shown in Figure 1 On the 5th day after irradiation, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital according to their weight, and blood was collected through the abdominal aorta to be killed. The whole brain was removed, and 3 mice in each group were used for pathological histological examination. The hippocampus and cerebral cortex of 5 mice were separated and stored at -80℃ for biochemical examination.

[0041] 2. Test indicators, results and analysis

[0042] 1. Histopathological evaluation (HE staining)

[0043] 1) The whole brain was fixed in 4% paraformaldehyde (Legen Biotechnology, Beijing) for at least 48 hours, then dehydrated, transparent, wax-impregnated, embedded and sliced. Histopathological study was performed using hematoxylin and eosin (H&E) staining. The results are shown in Figure 2 , the extent of the lesion was determined based on the results of histopathological staining, and the results were as follows;

[0044] Lesion severity: "+" indicates mild, "++" indicates mild, "+++" indicates moderate, and "++++" indicates severe.

[0045] 2) HE staining results showed that: in the control group, one case had focal neuronal nuclear enlargement in the hippocampal CA region, and chromatin dissolution 1+ / ++;

[0046] IR group: The arrangement of nerve cells was more disordered in 3 cases, focal nerve cell nuclei were enlarged in the CA region of the hippocampus, and chromatin was dissolved 2+++, 1+;

[0047] IR+Memantine group: 1 case had focal neuronal nuclear enlargement in the hippocampal CA region and chromatin dissolution 1+.

[0048] IR+HRW group: 1 case had focal neuronal nuclear enlargement in the CA region of the hippocampus with 1+ chromatin dissolution;

[0049] IR+Joint group: 1 case had focal neuronal nuclear enlargement in the hippocampal CA region and chromatin dissolution 1++ (n=3);

[0050] In general, the lesions in the CA region of the hippocampus were more severe in the IR group than in the control group, while there were no obvious abnormalities in the IR+Memantine group, IR+HRW group, and IR+Joint group. Memantine, HRW, and their combined effects can reduce radiation-induced brain damage.

[0051] 2. Effects of hydrogen-rich water, memantine and their combination on inflammatory factors and oxidative stress indicators

[0052] 1) Weigh an appropriate amount of brain tissue, homogenize and break it on ice, and use an ELISA reader (Thermo, Waltham, MA, USA) to detect SOD, MDA, GSH, and OH in the cerebral cortex according to the instructions of the kit. .- Level, see the results Figure 3 .

[0053] Superoxide dismutase (SOD) kit: Bio-Tech, S0101S;

[0054] Malondialdehyde (MDA) kit: Bio-Tech, S0131 S;

[0055] Reduced glutathione (GSH) kit: Nanjing Jiancheng, A006-2-1;

[0056] Hydroxyl radical (OH .- ) Kit: A018-1-1;

[0057] 2) From Figure 3 It can be seen that compared with the IR group, the control group and the IR+Mema group showed low MDA levels in the brain tissue (P<0.01), and the IR+HRW group and the IR+Jo int group also showed low MDA levels, but the differences were not statistically significant (P>0.05) ( Figure 3 a). Compared with the IR group, the levels of SOD and GSH in brain tissues of the other groups were higher, and the differences were statistically significant (P<0.05) ( Figure 3 b,3d). Compared with the IR group, the free radical scavenging ability of the Control group was increased (P<0.001), and there was no statistical difference in the other treatment groups (P>0.05) ( Figure 3c). It shows that hydrogen-rich water, memantine and their combination can improve the ability of the main antioxidant glutathione (GSH) and superoxide dismutase (SOD) in brain tissue to scavenge free radicals, and can inhibit malondialdehyde (MDA), a key byproduct of oxidation reactions. That is, in the brain tissue of rats with radiation-induced brain damage, memantine, hydrogen-rich water and their combination can inhibit the expression of inflammatory factors and improve oxidative stress.

[0058] 3. qRT-PCR detection of the effects of hydrogen-rich water, memantine and their combination on the mRNA expression levels of pyroptosis pathway genes in rats after ionizing radiation

[0059] 1) The expression of related genes NLRP3, NLRC4, ASC, Caspase1, Aim2, IL-18, and IL-1β was investigated. The operation method is as follows:

[0060] (1) Rats were killed 5 days after irradiation and their hippocampi were removed.

[0061] (2) Weigh 30-50 mg of hippocampus, add 1 ml of TRIzon, grind thoroughly, centrifuge, extract with chloroform, take the aqueous phase, precipitate with isopropanol, wash with ethanol, dry the precipitate, and then add 30 μL of RNase-Free Water to fully dissolve RNA to extract rat hippocampal RNA.

[0062] (3) RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit, and the gene expression level was detected according to the instructions of the SYBR Green qPCR Mix kit.

[0063] (4) Using GAPDH as the internal reference, the target gene was normalized and 2 -ΔΔCt The relative expression of NLRP3, NLRC4, ASC, Caspase1, Aim2, IL-18, and IL-1β genes in each group was compared by Figure 4 .

[0064] 2) Figure 4 The results showed that compared with the control group, the mRNA expression levels of NLRP3, NLRC4, and Caspase1 in the hippocampus of the IR group were higher, and the difference was statistically significant (P<0.05). The NLRC4 level and Aim2 level in the IR+Mema group, IR+HRW group, and IR+Joint group were lower than those in the IR group, and the difference was highly statistically significant (P<0.01). The Caspase1 level in the IR+Mema group was lower than that in the IR group, and the difference was statistically significant (P<0.05). The Caspase1 level in the IR+Mema group was lower than that in the IR group, and the difference was statistically significant (P<0.05).

[0065] The results showed that hydrogen-rich water, memantine and their combination could inhibit the expression of NLRC4, Caspase-1 and Aim2 genes in the hippocampus of rats with radiation-induced brain injury.

[0066] 4. Immunohistochemistry to detect NLRP3 and IL-1b protein expression

[0067] 1) Paraffin-embedded whole brain sections were dewaxed to water, antigen repaired, peroxidase inactivated, and blocked with goat blocking solution, and incubated with the detected antibodies overnight at 4°C, detected with GTVisionTM anti-mouse anti-rabbit immunohistochemistry kit (Gene Company, Shanghai, China), and imaged by microscope. IHC Profiler plug-in (https: / / sourceforge.net / projects / ihcprofiler) was used for quantitative immunohistochemical analysis. Paraffin-embedded lung tissue sections were dewaxed, rehydrated, antigen repaired, peroxidase inactivated, and blocked with goat blocking solution, and incubated with the detected antibodies overnight at 4°C, detected with GTVisionTM anti-mouse anti-rabbit immunohistochemistry kit (Gene Company, Shanghai, China), and scanned and imaged by CaseViewer. The results are shown in Figure 5 ; ImageJ software (https: / / imagej.nih.gov / ij / ) was used for quantitative immunohistochemical analysis, and the secreted protein IL-1β was analyzed according to the whole brain tissue section. Immunohistochemistry further verified and observed the intracellular localization of related proteins. The results are shown in Figure 6-7 .

[0068] 2) From Figure 5-7 It can be seen that the immunohistochemical results of NLRP3 showed that the expression of NLRP3 in CA1, CA3 and DG areas of the irradiated group increased, while the IR+Mema group, IR+HRW group and IR+joint group seemed to alleviate these changes (P<0.05); while the immunohistochemical results of IL-1β showed that the expression of IL-1β in the brain of the irradiated group increased, and the expression levels of the IR+Mema group, IR+HRW group and IR+joint group were lower, and the difference was statistically significant (P<0.05). The results further confirmed that memantine, hydrogen-rich water and their combined effects improved radiation-induced brain damage by blocking NLRP3-related pathways.

[0069] 5. Western blot detection of the effects of memantine, hydrogen-rich water and their combined effects on the expression of NLRP3, NLRC4, Caspase1, Cleaved Caspase1, ASC and IL-18 proteins in the hippocampus of rats with radiation-induced brain injury

[0070] 1) The expression of related proteins NLRP3, NLRC4, Caspase1, ASC, and IL-18 proteins was investigated. The operation method is as follows:

[0071] (1) Take 20 mg of hippocampal tissue from rats, extract tissue protein using a protein extraction kit, and use a BCA kit to measure protein concentration. Dilute the extracted protein 15 times, add 25 μL of diluted protein solution and 200 μL of BCA working solution (A:B=50:1) to a 96-well plate and react at 37°C for 30 min. Detect the absorbance at 562 nm using an ELISA reader, and calculate the sample concentration using the absorbance concentration calculation formula; use the sample with the lowest concentration as a reference to adjust the concentration of all samples to the same level.

[0072] (2) Sample loading: 6 μL per well;

[0073] (3) Electrophoresis: voltage 60 V, time about 2 h;

[0074] (4) Membrane transfer: Cut a strip of PVDF membrane of appropriate length and width, soak the membrane in methanol, and place the filter paper, membrane, gel, and filter paper in a "sandwich" style.

[0075] (5) Blocking: The membrane was blocked in 5% milk in TBST for 2 h;

[0076] (6) Incubation with primary antibodies: Add primary antibodies (NLRP3 (1:1800, Wanleibio, WL02635), NLRC4 (1:800, ThermoFisher, PA5-79739), Caspase-1 / Cleaved Caspase-1 (1:600, Wanleibio, WL3450), ASC (1:500, Wanleibio, WL02462) and GAPDH (1:1200, Servicebio, GB15004-100)) and incubate on a shaker at 4°C overnight.

[0077] (7) Cleaning: Rinse with TBST three times, 10 min each time

[0078] (8) Incubation with secondary antibodies: add HRP-labeled goat anti-mouse antibody and goat anti-rabbit antibody (dilution ratio of 1:5000) respectively and incubate at 37°C for 2 h;

[0079] (9) Washing: rinse with TBST three times, 10 min each time;

[0080] (10) Exposure: Mix the developer kit A and B in a ratio of 1:1. When using, cover the membrane and react for about 1 min. Place it in a chemiluminescence developer for development.

[0081] (11) Analysis: The grayscale values ​​of the protein bands were analyzed using Image J software. The internal reference protein was GAPDH, and the relative expression levels of the proteins were analyzed using the grayscale value ratios of NLRP3, NLRC4, Caspase1, ASC, and IL-18 to GAPDH. The results are shown in Figure 8-9 .

[0082] 2) Figure 8-9 The results showed that compared with the control group, the protein expression levels of NLRP3, NLRC4, ASC, Caspase1, CleavedCaspase1, and IL-18 in the IR group were higher, and the differences were statistically significant (P<0.05); compared with the IR group, the protein expression levels of NLRP3, NLRC4, CASP1, ASC, and IL-18 in the IR+Mema group, IR+HRW group, and IR+joint group were lower, except for the expression levels of NLRP3 in the IR+mema group and IR+HRW group, and the expression levels of ASC and Casp1 in the IR+mema group, which were not statistically different (P>0.05); compared with the IR group, the protein levels of NLRP3, CASP1, ASC, and IL-18 in the other groups were statistically significant (P<0.05). These findings indicate that Mema, HRW, and their combined effects improve brain damage induced by ionizing radiation by blocking the NLRP3, NLRC4, and Caspase1 pathways.

[0083] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition for treating radiation brain damage, characterized in that: The invention comprises an active ingredient a; the active ingredient a is memantine and its salt.

2. The pharmaceutical composition for treating radiation brain injury according to claim 1, characterized in that: The salts include hydrochlorides.

3. The pharmaceutical composition for treating radiation brain injury according to claim 1, characterized in that: It also includes an active ingredient b; the active ingredient b is hydrogen-rich water.

4. The pharmaceutical composition for treating radiation brain injury according to claim 3, characterized in that: The concentration of the hydrogen-rich water is greater than or equal to 0.5 mmol / L.

5. The pharmaceutical composition for treating radiation brain injury according to claim 3, characterized in that: Pharmaceutically acceptable adjuvants and / or excipients are also included.

6. A pharmaceutical preparation comprising the pharmaceutical composition according to any one of claims 1 to 5, characterized in that: These include tablets, capsules, powders, syrups, solutions, suspensions or aerosols.

7. Use of memantine and its salts or memantine and its salts in combination with hydrogen-rich water in the preparation of drugs for treating radiation-induced brain injury.

8. The use according to claim 7, characterized in that: Memantine and its salts or memantine and its salts combined with hydrogen-rich water can block the NLRP3, NLRC4, and Caspase-1 pathways.

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