Application of spermine in preparation of antidepressant

Abstract

The invention provides an application of spermine in preparation of an antidepressant, and belongs to the technical field of biological medicines. The spermine can act on a TREK-1 receptor, and activation of a human TREK-1 voltage-gated ion channel is remarkably inhibited. Experiments on model animal mice show that spermine treatment can enhance the chronic social contusion stress tolerance of the mice, and depression-like behaviors of the mice are remarkably reduced. In conclusion, spermine enhances the resistance of mice and other species to depression, can be developed into a potential antidepressant as a resource, and is applied to human and other animals to relieve depression. Secondly, spermine acts on a receptor TREK-1 in a targeting manner, and can be applied to research on related ion channel diseases and used as a potential therapeutic drug.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and in particular relates to the application of spermine in the preparation of antidepressants. Background Art

[0002] Spermine is an important polyamine with the chemical formula C 10 H 26 Spermidine is widely present in all eukaryotic cells and is involved in cell metabolism. It is synthesized from the amino acid ornithine and is considered to be one of the essential factors for cell growth. Currently, there are no reports on the effect of spermine on the perception of depression in humans and animals. Summary of the invention

[0003] The purpose of the present application is to provide the use of spermine in the preparation of antidepressants. Spermine can desensitize the induction of depression in humans or animals, thereby achieving an antidepressant effect.

[0004] The present application provides the use of spermine in the preparation of a drug for treating TREK-1 ion channel related diseases; the chemical structural formula of the spermine is shown in Formula I:

[0005]

[0006] Preferably, the TREK-1 ion channel-related diseases include one or more of depression, Alzheimer's disease and hypertension.

[0007] Preferably, when the TREK-1 ion channel-related disease is depression, the drug is used for at least one of the following aspects: 1) relieving depression; 2) assisting in relieving depression; 3) blunting the induction of depression in humans or animals; 4) enhancing the resistance of humans or animals to depression.

[0008] Preferably, the depression includes depression caused by social frustration.

[0009] The present application provides the use of spermine in preparing analgesic drugs.

[0010] Preferably, the drug is directed to humans or mammals.

[0011] Preferably, the dosage form of the drug includes an oral dosage form or an injection dosage form.

[0012] Preferably, spermine is the only active ingredient of the drug.

[0013] The present application provides the use of spermine in the preparation of a TREK-1 ion channel inhibitor; the chemical structural formula of the spermine is shown in Formula I:

[0014]

[0015] Preferably, spermine is the only active ingredient of the TREK-1 ion channel inhibitor.

[0016] The present application provides the use of spermine in the preparation of drugs for treating TREK-1 ion channel-related diseases. TREK-1 is inhibited as a receptor closely related to the perception of depression in the animal brain, and will blunt the animal's induction of depression for a certain period of time. Spermine can act on the TREK-1 receptor and significantly inhibit the activation of human TREK-1 voltage-gated ion channels. Experiments on model animals such as mice have shown that spermine treatment can enhance the tolerance of mice to chronic social frustration stress, and the depressive-like behavior of mice is significantly reduced. In summary, spermine enhances the resistance of species such as mice to depression, and can be developed as a resource into a potential antidepressant, which can be used in humans and other animals to relieve depression. Secondly, spermine targets the receptor TREK-1, which can be applied to the study of related ion channel diseases and as a potential therapeutic drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 The current and concentration effect curves of spermine inhibiting TREK-1; A is the current trajectory diagram of spermine inhibiting TREK-1 at different concentrations, and B is the simulation calculation of the half-inhibitory concentration of spermine;

[0019] Figure 2 The results of the behavioral test of sucrose preference experiment on mice using spermine showed that the sucrose preference of depressive mice increased;

[0020] Figure 3 This is a comparison of the effects of spermine on the content of monoamines in the mouse brain. DETAILED DESCRIPTION

[0021] The present application provides the use of spermine in the preparation of a drug for treating TREK-1 ion channel related diseases; the chemical structural formula of the spermine is shown in Formula I:

[0022]

[0023] In the specific implementation of the present application, the TREK-1 ion channel-related diseases include one or more of depression, Alzheimer's disease and hypertension.

[0024] In the specific implementation process of this application, the spermine was purchased from MedChemExpress (MCE).

[0025] In the specific implementation of this application, when the TREK-1 ion channel-related disease is depression, the drug is used for at least one of the following aspects:

[0026] 1) Relieve depression;

[0027] 2) Assist in relieving depression;

[0028] 3) blunting the sensitivity of humans or animals to depression;

[0029] 4) Enhance the resistance of humans or animals to depression.

[0030] TREK-1 is a type of pain-related voltage-gated calcium ion channel, which is expressed in large quantities in brain nerve cells, especially in the hippocampus. It is precisely because of the special distribution of TREK-1 that TREK-1 is found to be closely related to a variety of physiological and pathological functions of depression. In this application, spermine can target the receptor TREK-1 and can significantly inhibit the activation of TREK-1, so that humans and animals can be assisted by spermine to resist depression in the case of depression. In this application, spermine is a key chemical substance that relieves TREK-1-induced depression, so spermine can affect the perception of depression in humans and animals, passivate the induction of depression in humans or animals within a certain period of time (the experimental phase time is 7 days), and can be used to improve the resistance of animals to depression. This application verifies the effect of spermine on relieving depression in mice through a mouse depression-like model experiment. Synthesize TREK-1 plasmids of species such as mice and humans, and use patch clamp technology to verify that spermine inhibits the activation of mammalian TREK-1, confirming the universality of spermine in different species.

[0031] In the specific implementation of the present application, the depression includes depression caused by social frustration.

[0032] The present application also provides the use of spermine in preparing analgesic drugs.

[0033] In the specific implementation of the present application, the subjects of the drug include humans or mammals.

[0034] The present application does not specifically limit the dosage form of the drug, and conventional dosage forms in the art can be used. The present application preferably uses the drug orally or by injection to relieve depression in humans and animals.

[0035] In the specific implementation process of this application, spermine is the only active ingredient of the drug.

[0036] The present application also provides the use of spermine in the preparation of a TREK-1 ion channel inhibitor; the chemical structural formula of the spermine is shown in Formula I:

[0037]

[0038] In the specific implementation of this application, the spermine is the only active ingredient of the TREK-1 ion channel inhibitor.

[0039] In order to further illustrate the present application, the application of spermine provided by the present application in the preparation of antidepressants is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present application.

[0040] Example 1

[0041] Electrophysiological experiments on the targeting effects of spermine on hTREK-1 and other depression-related receptors

[0042] Preparation of hTREK-1 plasmid: The coding gene of TREK-1 protein was obtained by artificial synthesis, and inserted between HindIII and EcoR I of pEGFP-N1 vector (kanamycin resistance) to obtain a plasmid encoding TREK-1 protein; wherein, the accession information of the amino acid sequence of TREK-1 protein is: NCBI Reference Sequence: NP_001017424.1, and the accession information of the nucleotide sequence encoding TREK-1 protein is: Homo sapiens KCNK2 (Gene ID: 3776).

[0043] Plasmids such as hTREK-1 were transiently transfected and overexpressed in HEK293T cell lines. All HEK293T cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37° C. and 5% CO 2 .

[0044] Under an inverted microscope, cells with smooth cell membranes and uniform cytoplasm were selected, and patch clamp experiments were performed at room temperature of 20-25°C. WPI 0.86mm thin-walled borosilicate glass capillaries were selected as glass electrode materials. The glass electrodes were drawn on a drawing instrument (P-97, Shutter) in 5 steps. The diameter of the tip of the glass electrode was 1.5-3.0μm after thermal polishing. After drawing, the intracellular fluid was filled into the glass electrode. The initial resistance of the glass electrode was 1.5-2.5MΩ. After a high-impedance GΩ seal was formed between the electrode and the cell membrane, the fast capacitance of the electrode was compensated. Then a short and strong negative pressure was applied to quickly break the cell membrane clamped in the electrode, and then compensate the slow capacitance of the cell. After the whole-cell recording mode was formed, the cell was clamped to 0mV, and the cell was stabilized for 10min before the current was recorded using an appropriate pulse voltage (80 / -80mV). The drugs were perfused using BiolabRS200, and the switching speed between drugs was 50ms. The series resistance (Rs) was kept constant within the range of 5-8MΩ during the experiment, and the system series resistance compensation was generally between 30% and 60%. The experimental data were analyzed using PatchMaster software, and the data were further analyzed using Igor software. All results are expressed in the form of mean ± standard error (SEM), and n represents the number of experimental data.

[0045] The cells are slowly lifted by the electric micromanipulator system until they are flush with the bottom edge of the drug-adding port of the rapid drug-adding switching system (RSC-200). The drug-adding switching system is directly connected to the amplifier, and all experimental data are recorded in the computer software PatchMaster through the amplifier. Cells are stimulated and treated with drugs through PatchMaster and the switching drug-adding system, and the changes in channel current are recorded to detect the activity of different drugs on the channel. To ensure the accuracy of the experimental data, the sealing resistance and series resistance must be kept stable throughout the recording process.

[0046] TREK-1 channel external solution: 135mM NaCl, 5mM KCl, 1mM MgCl2, 1mM CaCl2, 5mM glucose and 10mM HEPES, adjusted to pH 7.3 with NaOH. The drugs used in the experiment need to be dissolved and prepared with the above electrode external solution.

[0047] TREK-1 channel internal solution: 150mM KCl, 1mM MgCl2, 1mM CaCl2, 5mM EGTA and 10mM HEPES, adjusted to pH 7.3 with KOH.

[0048] The results are as follows Figure 1 As shown in A, spermine inhibits the activation of hTREK-1 channels in a concentration-dependent manner.

[0049] Example 2

[0050] TREK-1 channel inhibitory activity of spermine

[0051] Based on the direct interaction between TREK-1 and spermine, whole-cell electrophysiological recordings were performed to verify the docking results.

[0052] TREK-1 channels were recorded using TREK-1 mode, and voltage-gated potassium channels were measured using a HEKA EPC10 amplifier with PatchMaster software. Glass electrodes were used for cell-attached recordings, with an input resistance of 3–5 MΩ. The perfusion tubes worked by gravity, and real-time data was sent back to the amplifier for processing via PatchMaster, and changes in current were recorded in real time. Extracellular solution for TREK-1 channels (mM): 135 NaCI, 5 KCI, 1 MgCl2, 1 CaCl2, 5 D-glucose, and 10 HEPES (pH 7.3–7.4 adjusted with NaOH); intracellular solution (mM): 150 KCI, 1 MgCl2, 5 EGTA, 10 HEPES (pH 7.3 adjusted with KOH), with a clamping voltage of -80 mV and a stimulation voltage of 30 mV. TREK channel extracellular solution (mM): 135NaCI, 5KCI, 1MgCl2, lCaCl2, 5D-glucose and 10HEPES (NaOH adjusted to pH 7.3); intracellular solution (mM): 150KCI, 1MgCl2, 5EGTA and 10HEPES (KOH adjusted to pH 7.3), TREK channel was recorded using Ramp mode, and the voltage gradually increased from -120mV to 60mV; HEK293T cells transfected with TREK-1 plasmid had a clamping voltage of -80mV, a duration of 50ms, and an excitation voltage of 0mV, with a duration of 100ms.

[0053] The results showed that 10 μM spermine almost completely inhibited the TREK-1 channel current, while 2.5 μM spermine inhibited the channel current by 44.50%. Spermine inhibited the TREK-1 channel in a concentration-dependent manner, with a half-inhibitory concentration of 3.07 μM. The results are shown in Figure 1 B in.

[0054] Example 3

[0055] Antidepressant activity of spermine

[0056] Depression induced by chronic social frustration stress model: Several non-breeding CD-1 male mice over 6 months old were purchased and kept in single cages for 7 days before the experiment to adapt to the environment. (Single cages are used to cultivate and maintain the aggressiveness of CD-1 mice. CD-1 mice can also be placed with two female mice for 3 to 4 days before being taken out to cultivate their aggressiveness).

[0057] C57BL / 6J mice (optimally aged 6 to 7 weeks) were purchased and adapted to the mouse room environment in advance for subsequent depression modeling experiments.

[0058] In the social defeat phase (experimental time is 7 to 10 days), the social defeat experimental cage (including bedding) was prepared one day in advance, and the CD-1 mice were placed in a cage on one side of the isolation baffle the night before. Experimental group: For the C57BL / 6J mice tested, they were required to undergo a social defeat stress experiment for 7 to 10 consecutive days, lasting 5 to 10 minutes each day. Every day, the C57BL / 6J mice were placed directly in the experimental cage containing CD-1 mice on the prepared side to experience 5 to 10 minutes of social confrontation. This process is called direct contact stress. Afterwards, the C57BL / 6J mice were placed on the other side of the isolation baffle of the experimental cage and left there for 24 hours as indirect psychological stress.

[0059] Note: The experimental process should be closely observed to ensure that high-quality aggressive behavior occurs normally. High-quality aggressive behavior must meet the requirements of continuous and periodic aggressive behavior within 5 to 10 minutes of the experiment; there must be no less than 1 round of attack per minute, and each round lasts about 5 to 10 seconds. If the two animals groom each other, the experiment is considered to have failed at this stage. The experimental process should be closely observed to avoid physical conflict between the two mice. After the 7-10 day social frustration experiment, all the tested C57BL / 6J mice were housed separately, and the subsequent behavioral testing phase was carried out 24 hours later (or according to the experimental design time).

[0060] Three days before the end of the depression model induction, the mice were allowed to eat freely. On the first day of the experiment, the mice were given a bottle of 1% sucrose water and a bottle of drinking water. On the second day, the two bottles were swapped to adapt to the sugar water. On the third day, the mice were fasted and deprived of water. On the fourth day, a bottle of sugar water and a bottle of drinking water were weighed and placed in each cage. After two hours, the amount of remaining water was weighed again. The difference between the two times was the amount of drinking water and sucrose water. Finally, the sugar water preference was calculated as sucrose water / (drinking water + sucrose water)*100%.

[0061] The above-mentioned mouse-related behavioral experiments show that intraperitoneal injection of spermine can help mice relieve depression-like behaviors. Spermine enhances the resistance of mice to depression and can be used as a resource to develop into a potential analgesic for use in humans and other animals to relieve depression. Secondly, spermine targets the receptor TREK-1 and can be used in the study of related ion channel diseases and as a potential therapeutic drug. Results can be found in Figure 2 and Figure 3 .

[0062] Although the above-mentioned embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present application.

Claims

1. Application of spermine in the preparation of a drug for treating TREK-1 ion channel related diseases; the chemical structural formula of the spermine is shown in Formula I:

2. The use according to claim 1, characterized in that: The TREK-1 ion channel related diseases include one or more of depression, Alzheimer's disease and hypertension.

3. The use according to claim 2, characterized in that: When the TREK-1 ion channel-related disease is depression, the drug is used for at least one of the following aspects: 1) Relieve depression; 2) Assist in relieving depression; 3) blunting the sensitivity of humans or animals to depression; 4) Enhance the resistance of humans or animals to depression.

4. The use according to claim 2, characterized in that: The depression includes depression caused by social frustration.

5. Application of spermine in the preparation of analgesic drugs.

6. The use according to any one of claims 1 to 5, characterized in that: The subjects of the drug include humans or mammals.

7. The use according to any one of claims 1 to 5, characterized in that: The dosage form of the drug includes an oral dosage form or an injection dosage form.

8. The use according to any one of claims 1 to 5, characterized in that: The spermine is the only active ingredient of the drug.

9. Use of spermine in the preparation of a TREK-1 ion channel inhibitor; the chemical structural formula of the spermine is shown in Formula I:

10. The use according to claim 9, characterized in that: The spermine is the only active ingredient of the TREK-1 ion channel inhibitor.

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